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

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.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.001317 ◽

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.

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Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽

10.1099/mic.0.001087 ◽

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.

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Roles for phthiocerol dimycocerosate lipids in Mycobacterium tuberculosis pathogenesis

Microbiology ◽

10.1099/mic.0.001042 ◽

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.

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2-Alkyl-4-quinolone quorum sensing molecules are biomarkers for culture-independent Pseudomonas aeruginosa burden in adults with cystic fibrosis

Journal of Medical Microbiology ◽

10.1099/jmm.0.001420 ◽

2021 ◽

Vol 70 (10) ◽

Author(s):

Nur Masirah M. Zain ◽

Karmel Webb ◽

Iain Stewart ◽

Nigel Halliday ◽

David A. Barrett ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Type Species ◽

Bacterial Load ◽

Pulmonary Exacerbation ◽

Content Type ◽

Link Type ◽

Culture Independent ◽

Clinical Stability

Introduction. Pseudomonas aeruginosa produces quorum sensing signalling molecules including 2-alkyl-4-quinolones (AQs), which regulate virulence factor production in the cystic fibrosis (CF) airways. Hypothesis/Gap statement. Culture can lead to condition-dependent artefacts which may limit the potential insights and applications of AQs as minimally-invasive biomarkers of bacterial load. Aim. We aimed to use culture-independent methods to explore the correlations between AQ levels and live P. aeruginosa load in adults with CF. Methodology. Seventy-five sputum samples at clinical stability and 48 paired sputum samples obtained at the beginning and end of IV antibiotics for a pulmonary exacerbation in adults with CF were processed using a viable cell separation technique followed by quantitative P. aeruginosa polymerase chain reaction (qPCR). Live P. aeruginosa qPCR load was compared with the concentrations of three AQs (HHQ, NHQ and HQNO) detected in sputum, plasma and urine. Results. At clinical stability and the beginning of IV antibiotics for pulmonary exacerbation, HHQ, NHQ and HQNO measured in sputum, plasma and urine were consistently positively correlated with live P. aeruginosa qPCR load in sputum, compared to culture. Following systemic antibiotics live P. aeruginosa qPCR load decreased significantly (P<0.001) and was correlated with a reduction in plasma NHQ (plasma: r=0.463, P=0.003). Conclusion. In adults with CF, AQ concentrations correlated more strongly with live P. aeruginosa bacterial load measured by qPCR compared to traditional culture. Prospective studies are required to assess the potential of systemic AQs as biomarkers of P. aeruginosa bacterial burden.

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

Microbial Genomics ◽

10.1099/mgen.0.000505 ◽

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.

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

Microbiology ◽

10.1099/mic.0.001035 ◽

2021 ◽

Vol 167 (2) ◽

Cited By ~ 1

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.

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Corynebacterium striatum thrombophlebitis: a nosocomial multidrug-resistant disease?

Access Microbiology ◽

10.1099/acmi.0.000307 ◽

2021 ◽

Vol 3 (12) ◽

Author(s):

Julie Tang ◽

Dimitri Kornblum ◽

Nagisa Godefroy ◽

Gentiane Monsel ◽

Jérome Robert ◽

...

Keyword(s):

Type Species ◽

Bacterial Load ◽

Anticoagulation Therapy ◽

Specific Treatment ◽

Multidrug Resistant ◽

Septic Thrombophlebitis ◽

Content Type ◽

Link Type ◽

Wide Range ◽

Corynebacterium Striatum

Introduction. Corynebacterium striatum is a non-Diphteriae commensal bacterium with a wide range of pathogenicity. The identification of multidrug-resistant (MDR) C. striatum is concerning because drug susceptibility testing is not usually performed in microbiology laboratories. There is no consensus yet on the treatment of septic thrombophlebitis in this situation. Case report. We report here the first case of a quinquagenarian patient with a history of AIDS and fungic endocarditis, who was diagnosed with a nosocomial thrombophlebitis in the right jugular vein caused by C. striatum . Bitherapy with daptomycin for 12 days and linezolid for 23 days was combined with a therapeutic anticoagulant. The follow-up included weekly cervical ultrasound controls. The efficiency of the treatment and the stability of the lesions allowed us to alleviate the medication with a prophylactic dose of anticoagulant. The patient was discharged from hospital and showed no signs of recurrence after 12 months. Conclusion. The lack of consensus relative to the management of septic thrombophlebitis precludes the validation of a specific treatment for the condition. Our results suggest that a combination that includes removal of the medical device is needed. A total of 6 weeks of antibiotherapy should be applied, starting with 2 weeks of vancomycin or a combination of antibiotitherapy with daptomycin in order to reduce the bacterial load and avoid resistance. Six weeks of anticoagulation therapy is effective.

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Halovivax limisalsi sp. nov., an extremely halophilic archaeon from hypersaline mud

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.063297-0 ◽

2014 ◽

Vol 64 (Pt_10) ◽

pp. 3422-3426 ◽

Cited By ~ 10

Author(s):

Mohammad Ali Amoozegar ◽

Ali Makhdoumi-Kakhki ◽

Maliheh Mehrshad ◽

Siavash Riazi ◽

Antonio Ventosa

Keyword(s):

Type Species ◽

Optimal Growth ◽

Rpob Gene ◽

Hypersaline Lake ◽

Rrna Gene ◽

Halophilic Archaeon ◽

Content Type ◽

Link Type ◽

Extremely Halophilic Archaeon ◽

Ph Range

A Gram-stain-negative, cream‐pigmented, motile, extremely halophilic archaeon, designated strain IC38T, was isolated from a saline mud sample taken from a hypersaline lake, Aran-Bidgol, in Iran. The strain required at least 2.5 M NaCl for growth. However, MgCl2 was not required. Optimal growth occurred with 4.3 M NaCl and 0.2 M MgCl2. The optimum pH and temperature for growth were pH 7.0 and 35 °C, respectively, and strain IC38T was able to grow over a pH range of 6.5–9.0, and a temperature range of 25–45 °C. Analysis of the 16S rRNA gene sequence revealed that strain IC38T clustered with the two species of the genus Halovivax , Halovivax asiaticus EJ-46T and Halovivax ruber XH-70T, with sequence similarities of 96.4 % and 96.1 %, respectively. The similarities between the rpoB′ gene of the novel strain and Halovivax asiaticus and Halovivax ruber were 90.7 % and 90.3 %, respectively. The polar lipid pattern of strain IC38T consisted of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Three unidentified glycolipids and two minor phospholipids were also observed. The DNA G+C content of strain IC38T was 62.6 mol%. On the basis of the phylogenetic analysis, as well as the biochemical and physiological characteristics, the new isolate is suggested to be a representative of a novel species of the genus Halovivax , for which the name Halovivax limisalsi sp. nov. is proposed. The type strain of Halovivax limisalsi is IC38T ( = IBRC-M 10022T = KCTC 4051T).

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

Microbial Genomics ◽

10.1099/mgen.0.000437 ◽

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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Genome analyses of 174 strains of Mycobacterium tuberculosis provide insight into the evolution of drug resistance and reveal potential drug targets

Microbial Genomics ◽

10.1099/mgen.0.000542 ◽

2021 ◽

Vol 7 (3) ◽

Author(s):

Helianthous Verma ◽

Shekhar Nagar ◽

Shivani Vohra ◽

Shubhanshu Pandey ◽

Devi Lal ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Target ◽

Drug Targets ◽

Type Species ◽

Close Association ◽

Drug Resistant ◽

Potential Drug ◽

Sequence Alignments ◽

Content Type ◽

Link Type

Mycobacterium tuberculosis is a known human pathogen that causes the airborne infectious disease tuberculosis (TB). Every year TB infects millions of people worldwide. The emergence of multi-drug resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis strains against the first- and second-line anti-TB drugs has created an urgent need for the development and implementation of new drug strategies. In this study, the complete genomes of 174 strains of M. tuberculosis are analysed to understand the evolution of molecular drug target (MDT) genes. Phylogenomic placements of M. tuberculosis strains depicted close association and temporal clustering. Selection pressure analysis by deducing the ratio of non-synonymous to synonymous substitution rates (dN/dS) in 51 MDT genes of the 174 M . tuberculosis strains led to categorizing these genes into diversifying (D, dN/dS>0.70), moderately diversifying (MD, dN/dS=0.35–0.70) and stabilized (S, dN/dS<0.35) genes. The genes rpsL, gidB, pncA and ahpC were identified as diversifying, and Rv0488, kasA, ndh, ethR, ethA, embR and ddn were identified as stabilized genes. Furthermore, sequence similarity networks were drawn that supported these divisions. In the multiple sequence alignments of diversifying and stabilized proteins, previously reported resistance mutations were checked to predict sensitive and resistant strains of M. tuberculosis . Finally, to delineate the potential of stabilized or least diversified genes/proteins as anti-TB drug targets, protein–protein interactions of MDT proteins with human proteins were analysed. We predict that kasA (dN/dS=0.29), a stabilized gene that encodes the most host-interacting protein, KasA, should serve as a potential drug target for the treatment of TB.

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