scholarly journals Revisiting the methods for detecting Mycobacterium tuberculosis: what has the new millennium brought thus far?

2021 ◽  
Vol 3 (8) ◽  
Author(s):  
Thales Alves Campelo ◽  
Paulo Rafael Cardoso de Sousa ◽  
Lucas de Lima Nogueira ◽  
Cristiane Cunha Frota ◽  
Paulo Renato Zuquim Antas
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Low Cost ◽  
Clinical Specimen ◽  
Simultaneous Detection ◽  
Smear Microscopy ◽  
Sensitivity Testing ◽  
Gold Standard Method ◽  
Content Type ◽  
Link Type

Tuberculosis (TB) affects around 10 million people worldwide in 2019. Approximately 3.4 % of new TB cases are multidrug-resistant. The gold standard method for detecting Mycobacterium tuberculosis , which is the aetiological agent of TB, is still based on microbiological culture procedures, followed by species identification and drug sensitivity testing. Sputum is the most commonly obtained clinical specimen from patients with pulmonary TB. Although smear microscopy is a low-cost and widely used method, its sensitivity is 50–60 %. Thus, owing to the need to improve the performance of current microbiological tests to provide prompt treatment, different methods with varied sensitivity and specificity for TB diagnosis have been developed. Here we discuss the existing methods developed over the past 20 years, including their strengths and weaknesses. In-house and commercial methods have been shown to be promising to achieve rapid diagnosis. Combining methods for mycobacterial detection systems demonstrates a correlation of 100 %. Other assays are useful for the simultaneous detection of M. tuberculosis species and drug-related mutations. Novel approaches have also been employed to rapidly identify and quantify total mycobacteria RNA, including assessments of global gene expression measured in whole blood to identify the risk of TB. Spoligotyping, mass spectrometry and next-generation sequencing are also promising technologies; however, their cost needs to be reduced so that low- and middle-income countries can access them. Because of the large impact of M. tuberculosis infection on public health, the development of new methods in the context of well-designed and -controlled clinical trials might contribute to the improvement of TB infection control.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Nirbhay Singh ◽  
Anu Chauhan ◽  
Ram Kumar ◽  
Sudheer Kumar Singh
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Low Ph ◽  
Stress Conditions ◽  
Starvation Stress ◽  
Content Type ◽  
E Coli ◽  
Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

Mycobacterium parakoreense sp. nov., a slowly growing non-chromogenic species related to Mycobacterium koreense , isolated from a human clinical specimen

2013 ◽  
Vol 63 (Pt_6) ◽  
pp. 2301-2308 ◽  
Author(s):  
Byoung-Jun Kim ◽  
Seok-Hyun Hong ◽  
Hee-Kyung Yu ◽  
Young-Gil Park ◽  
Joseph Jeong ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Sputum Sample ◽  
Clinical Specimen ◽  
Mycolic Acid ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

A previously undescribed, slowly growing, non-chromogenic Mycobacterium strain (299T) was isolated from the sputum sample of a patient with a symptomatic pulmonary infection. Phenotypically, strain 299T was generally similar to Mycobacterium koreense DSM 45576T and Mycobacterium triviale ATCC 23292T. The 16S rRNA gene sequence of strain 299T was similar to that of M. koreense DSM 45576T (GenBank accession no. AY734996, 99.5 % similarity); however, it differed substantially from that of M. triviale ATCC 23292T (X88924, 98.2 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 299T clustered together with M. koreense DSM 45576T and M. triviale ATCC 23292T, supported by high bootstrapping values (99 %). Unique mycolic acid profiles and phylogenetic analysis based on two different chronometer molecules, the hsp65 and rpoB genes, strongly supported the taxonomic status of this strain as representing a distinct species. These data support the conclusion that strain 299T represents a novel mycobacterial species, for which the name Mycobacterium parakoreense sp. nov. is proposed. The type strain is 299T ( = DSM 45575T = KCTC 19818T).

scholarly journals Roles for phthiocerol dimycocerosate lipids in Mycobacterium tuberculosis pathogenesis

Microbiology ◽  
2021 ◽  
Author(s):  
Céline Rens ◽  
Joseph D. Chao ◽  
Danielle L. Sexton ◽  
Elitza I. Tocheva ◽  
Yossef Av-Gay
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Cell Envelope ◽  
Infectious Agent ◽  
Resistant Bacteria ◽  
Content Type ◽  
Link Type ◽  
Drug Resistant Bacteria ◽  
Macrophage Phagocytosis ◽  
Key Steps

The success of Mycobacterium tuberculosis as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and extensively drug-resistant bacteria has renewed global concerns about this pathogen and understanding its virulence strategies will be essential in the fight against tuberculosis. The current review will focus on phthiocerol dimycocerosates (PDIMs), a long-known and well-studied group of complex lipids found in the M. tuberculosis cell envelope. Numerous studies show a role for PDIMs in several key steps of M. tuberculosis pathogenesis, with recent studies highlighting its involvement in bacterial virulence, in association with the ESX-1 secretion system. Yet, the mechanisms by which PDIMs help M. tuberculosis to control macrophage phagocytosis, inhibit phagosome acidification and modulate host innate immunity, remain to be fully elucidated.

Rifampin-resistance-associated mutations in the rifampin-resistance-determining region of the rpoB gene of Mycobacterium tuberculosis clinical isolates in Shanghai, PR China

2021 ◽  
Author(s):  
Yinjuan Guo ◽  
Xingwei Cao ◽  
Jinghui Yang ◽  
Xiaocui Wu ◽  
Yin Liu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Mutation Frequency ◽  
Rpob Gene ◽  
Clinical Isolates ◽  
Content Type ◽  
Link Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Rifampin Resistance

Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the β-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive. Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance. Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis . Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis. Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01). Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.

scholarly journals Pathogenomic analyses of Mycobacterium microti, an ESX-1-deleted member of the Mycobacterium tuberculosis complex causing disease in various hosts

2021 ◽  
Vol 7 (2) ◽  
Author(s):  
Mickael Orgeur ◽  
Wafa Frigui ◽  
Alexandre Pawlik ◽  
Simon Clark ◽  
Ann Williams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pig ◽  
Type Species ◽  
Clinical Isolates ◽  
Recent Common Ancestor ◽  
Genome Sequences ◽  
Content Type ◽  
Link Type ◽  
Vaccine Potential ◽  
Mycobacterium Microti

Mycobacterium microti is an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC), which was originally isolated from voles, but has more recently also been isolated from other selected mammalian hosts, including occasionally from humans. Here, we have generated and analysed the complete genome sequences of five representative vole and clinical M. microti isolates using PacBio- and Illumina-based technologies, and have tested their virulence and vaccine potential in SCID (severe combined immune deficient) mouse and/or guinea pig infection models. We show that the clinical isolates studied here cluster separately in the phylogenetic tree from vole isolates and other clades from publicly available M. microti genome sequences. These data also confirm that the vole and clinical M. microti isolates were all lacking the specific RD1mic region, which in other tubercle bacilli encodes the ESX-1 type VII secretion system. Biochemical analysis further revealed marked phenotypic differences between isolates in type VII-mediated secretion of selected PE and PPE proteins, which in part were attributed to specific genetic polymorphisms. Infection experiments in the highly susceptible SCID mouse model showed that the clinical isolates were significantly more virulent than the tested vole isolates, but still much less virulent than the M. tuberculosis H37Rv control strain. The strong attenuation of the ATCC 35872 vole isolate in immunocompromised mice, even compared to the attenuated BCG (bacillus Calmette–Guérin) vaccine, and its historic use in human vaccine trials encouraged us to test this strain’s vaccine potential in a guinea pig model, where it demonstrated similar protective efficacy as a BCG control, making it a strong candidate for vaccination of immunocompromised individuals in whom BCG vaccination is contra-indicated. Overall, we provide new insights into the genomic and phenotypic variabilities and particularities of members of an understudied clade of the MTBC, which all share a recent common ancestor that is characterized by the deletion of the RD1mic region.

scholarly journals Ribosome hibernation: a new molecular framework for targeting nonreplicating persisters of mycobacteria

Microbiology ◽  
2021 ◽  
Vol 167 (2) ◽  
Author(s):  
Yunlong Li ◽  
Manjuli R. Sharma ◽  
Ravi K. Koripella ◽  
Nilesh K. Banavali ◽  
Rajendra K. Agrawal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Drug Regimen ◽  
Content Type ◽  
Link Type ◽  
A Minor ◽  
Molecular Framework

Treatment of tuberculosis requires a multi-drug regimen administered for at least 6 months. The long-term chemotherapy is attributed in part to a minor subpopulation of nonreplicating Mycobacterium tuberculosis cells that exhibit phenotypic tolerance to antibiotics. The origins of these cells in infected hosts remain unclear. Here we discuss some recent evidence supporting the hypothesis that hibernation of ribosomes in M. tuberculosis, induced by zinc starvation, could be one of the primary mechanisms driving the development of nonreplicating persisters in hosts. We further analyse inconsistencies in previously reported studies to clarify the molecular principles underlying mycobacterial ribosome hibernation.

scholarly journals Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction

2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Daniela Gaio ◽  
Kay Anantanawat ◽  
Joyce To ◽  
Michael Liu ◽  
Leigh Monahan ◽  
...  
Keyword(s):  
High Throughput ◽  
Type Species ◽  
De Novo ◽  
Low Cost ◽  
Simple Modification ◽  
Content Type ◽  
Library Construction ◽  
Link Type ◽  
Construction Methods ◽  
Research Programmes

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

scholarly journals Status of rpoB gene mutation associated with rifampicin-resistant Mycobacterium tuberculosis isolated in a rural setting in Nepal

2021 ◽  
Author(s):  
Saroj Adhikari ◽  
Bhuvan Saud ◽  
Sunil Sunar ◽  
Sheshraj Ghimire ◽  
Bhawani Prasad Yadav
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Bacterial Load ◽  
Latent Tuberculosis ◽  
Rpob Gene ◽  
Rural Setting ◽  
Molecular Technique ◽  
Content Type ◽  
Link Type

Mycobacterium tuberculosis ranks among the top 10 causes of deaths in Nepal despite the country having a long history of national tuberculosis prevention programmes that have proved very successful in the control of tuberculosis. Several cases of active or latent tuberculosis are still missing despite that the number of infected individuals is increasing each year. Microscopy has its own limitations and factors like low bacterial load, quality of sample, quality of smear, experience of microscopist etc. influence the overall sensitivity of the test. The implementation of a molecular technique-based rapid, point-of-care testing system offers higher sensitivity in the early diagnosis of tuberculosis. Cepheid GeneXpert is the most commonly used molecular technology in Nepal. It is a cartridge-based semi-quantitative, nested real-time PCR-based diagnostic system. It detects mutations in the beta-subunit of RNA polymerase (rpoB) gene that lead to rifampicin resistance (RR) in M. tuberculosis complex. The present study aims to increase our understanding of the epidemiology of mutations in the rpoB gene in tuberculosis-positive patients by using the Xpert MTB/RIF assay in a rural setting in Pyuthan Hospital, Nepal. Sputum from 2733 patients was tested for the diagnosis of tuberculosis using the Cepheid GeneXpert system between July 2018 and January 2020 at Pyuthan Hospital. Two hundred and ninety-seven of these samples (10.86 %) were positive for M. tuberculosis , of which 3.3 % (10/297) were rifampicin-resistant. Among rifampicin-resistant tuberculosis (RR-TB) patients, 50.0 % (5/10) showed mutations located in codons 529–533 (probe E) of the rpoB gene, followed by others. The GeneXpert system can be a convenient, highly sensitive, rapid and accurate tool for the diagnosis of tuberculosis, also identifying RR-TB and at the same time determining the molecular epidemiology of rifampin resistance-associated mutations in rural and/or resource-limited laboratory settings.

scholarly journals Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

2020 ◽  
Vol 6 (7) ◽  
Author(s):  
Erin P. Price ◽  
Valentina Soler Arango ◽  
Timothy J. Kidd ◽  
Tamieka A. Fraser ◽  
Thuy-Khanh Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Type Species ◽  
Simultaneous Detection ◽  
Diagnostic Methods ◽  
Achromobacter Xylosoxidans ◽  
Resistant Bacteria ◽  
Pcr Assay ◽  
Content Type ◽  
Link Type

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

scholarly journals Mycobacterium tuberculosis complex lineage 5 exhibits high levels of within-lineage genomic diversity and differing gene content compared to the type strain H37Rv

2021 ◽  
Vol 7 (7) ◽  
Author(s):  
C. N'Dira Sanoussi ◽  
Mireia Coscolla ◽  
Boatema Ofori-Anyinam ◽  
Isaac Darko Otchere ◽  
Martin Antonio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Reference Genome ◽  
De Novo ◽  
Genomic Diversity ◽  
Gene Content ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Content Type ◽  
Link Type

Pathogens of the Mycobacterium tuberculosis complex (MTBC) are considered to be monomorphic, with little gene content variation between strains. Nevertheless, several genotypic and phenotypic factors separate strains of the different MTBC lineages (L), especially L5 and L6 (traditionally termed Mycobacterium africanum ) strains, from each other. However, this genome variability and gene content, especially of L5 strains, has not been fully explored and may be important for pathobiology and current approaches for genomic analysis of MTBC strains, including transmission studies. By comparing the genomes of 355 L5 clinical strains (including 3 complete genomes and 352 Illumina whole-genome sequenced isolates) to each other and to H37Rv, we identified multiple genes that were differentially present or absent between H37Rv and L5 strains. Additionally, considerable gene content variability was found across L5 strains, including a split in the L5.3 sub-lineage into L5.3.1 and L5.3.2. These gene content differences had a small knock-on effect on transmission cluster estimation, with clustering rates influenced by the selected reference genome, and with potential overestimation of recent transmission when using H37Rv as the reference genome. We conclude that full capture of the gene diversity, especially high-resolution outbreak analysis, requires a variation of the single H37Rv-centric reference genome mapping approach currently used in most whole-genome sequencing data analysis pipelines. Moreover, the high within-lineage gene content variability suggests that the pan-genome of M. tuberculosis is at least several kilobases larger than previously thought, implying that a concatenated or reference-free genome assembly (de novo) approach may be needed for particular questions.

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