scholarly journals Correlates between Models of Virulence for Mycobacterium tuberculosis among Isolates of the Central Asian Lineage: a Case for Lysozyme Resistance Testing?

2015 ◽  
Vol 83 (6) ◽  
pp. 2213-2223 ◽  
Author(s):  
Claire Pardieu ◽  
Nicola Casali ◽  
Simon O. Clark ◽  
Richard Hooper ◽  
Ann Williams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Resistance Testing ◽  
Snp Analysis ◽  
Single Nucleotide ◽  
Content Type ◽  
Differential Outcomes ◽  
Central Asian ◽  
Strain H37rv

Virulence factors (VFs) contribute to the emergence of new humanMycobacterium tuberculosisstrains, are lineage dependent, and are relevant to the development ofM. tuberculosisdrugs/vaccines. VFs were sought withinM. tuberculosislineage 3, which has the Central Asian (CAS) spoligotype. Three isolates were selected from clusters previously identified as dominant in London, United Kingdom. Strain-associated virulence was studied in guinea pig, monocyte-derived macrophage, and lysozyme resistance assays. Whole-genome sequencing, single nucleotide polymorphism (SNP) analysis, and a literature review contributed to the identification of SNPs of interest. The animal model revealed borderline differences in strain-associated pathogenicity.Ex vivo, isolate C72 exhibited statistically significant differences in intracellular growth relative to C6 and C14. SNP candidates inducing lower fitness levels included 123 unique nonsynonymous SNPs, including three located in genes (lysX,caeA, andponA2) previously identified as VFs in the laboratory-adapted reference strain H37Rv and shown to confer lysozyme resistance. C72 growth was most affected by lysozymein vitro. A BLAST search revealed that all three SNPs of interest (C35F, P76Q, and P780R) also occurred in Tiruvallur, India, and in Uganda. Unlike C72, however, no single isolate identified through BLAST carried all three SNPs simultaneously. CAS isolates representative of three medium-sized human clusters demonstrated differential outcomes in models commonly used to estimate strain-associated virulence, supporting the idea that virulence varies within, not just across,M. tuberculosislineages. Three VF SNPs of interest were identified in two additional locations worldwide, which suggested independent selection and supported a role for these SNPs in virulence. The relevance of lysozyme resistance to strain virulence remains to be established.

scholarly journals Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Jichan Jang ◽  
Ryangyeo Kim ◽  
Minjeong Woo ◽  
Jinsun Jeong ◽  
Da Eun Park ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Ex Vivo ◽  
Drug Candidate ◽  
Content Type ◽  
Tb Treatment ◽  
Pump Activity ◽  
Efflux Pump Inhibitors ◽  
Combinatorial Strategy

ABSTRACT New and improved treatments for tuberculosis (TB) are urgently needed. Recently, it has been demonstrated that verapamil, an efflux inhibitor, can reduce bacterial drug tolerance caused by efflux pump activity when administered in combination with available antituberculosis agents. The aim of this study was to evaluate the effectiveness of verapamil in combination with the antituberculosis drug candidate Q203, which has recently been developed and is currently under clinical trials as a potential antituberculosis agent. We evaluated changes in Q203 activity in the presence and absence of verapamil in vitro using the resazurin microplate assay and ex vivo using a microscopy-based phenotypic assay for the quantification of intracellular replicating mycobacteria. Verapamil increased the potency of Q203 against Mycobacterium tuberculosis both in vitro and ex vivo, indicating that efflux pumps are associated with the activity of Q203. Other efflux pump inhibitors also displayed an increase in Q203 potency, strengthening this hypothesis. Therefore, the combination of verapamil and Q203 may be a promising combinatorial strategy for anti-TB treatment to accelerate the elimination of M. tuberculosis.

scholarly journals Phenotypically Adapted Mycobacterium tuberculosis Populations from Sputum Are Tolerant to First-Line Drugs

2016 ◽  
Vol 60 (4) ◽  
pp. 2476-2483 ◽  
Author(s):  
Obolbek Turapov ◽  
Benjamin D. O'Connor ◽  
Asel A. Sarybaeva ◽  
Caroline Williams ◽  
Hemu Patel ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liquid Medium ◽  
Culture Supernatant ◽  
Ex Vivo ◽  
First Line ◽  
Content Type ◽  
Host Environment ◽  
Solid Media ◽  
Bactericidal Effects

ABSTRACTTuberculous sputum contains multipleMycobacterium tuberculosispopulations with different requirements for isolationin vitro. These include cells that form colonies on solid media (plateableM. tuberculosis), cells requiring standard liquid medium for growth (nonplateableM. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependentM. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of theseM. tuberculosispopulations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10reductions), and SN-dependentM. tuberculosiswas more tolerant to streptomycin and isoniazid than the plateable and nonplateableM. tuberculosisstrains. Susceptibility of plateableM. tuberculosisto bactericidal drugs was significantly increased after passagein vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simpleex vivosystem for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.

scholarly journals Heterologous Production of 1-Tuberculosinyladenosine in Mycobacterium kansasii Models Pathoevolution towards the Transcellular Lifestyle of Mycobacterium tuberculosis

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Marwan Ghanem ◽  
Jean-Yves Dubé ◽  
Joyce Wang ◽  
Fiona McIntosh ◽  
Daniel Houle ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Virulence Factors ◽  
Ex Vivo ◽  
Nontuberculous Mycobacterium ◽  
Specific Gene ◽  
Bacterial Survival ◽  
Mycobacterium Kansasii ◽  
Content Type ◽  
Lipid Species

ABSTRACT Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24 h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and Ccr2−/− mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation. IMPORTANCE This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to M. tuberculosis. On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen.

scholarly journals Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant MycobacteriaIn VitroandEx Vivowithin Macrophages

2015 ◽  
Vol 59 (12) ◽  
pp. 7693-7699 ◽  
Author(s):  
David Pires ◽  
Emília Valente ◽  
Marta Filipa Simões ◽  
Nuno Carmo ◽  
Bernard Testa ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Acquired Resistance ◽  
Point Mutations ◽  
Intrinsic Activity ◽  
Significant Activity ◽  
Content Type ◽  
Long Chain ◽  
Pyrazinoic Acid

ABSTRACTPyrazinamide (PZA) is active against majorMycobacterium tuberculosisspecies (M. tuberculosis,M. africanum, andM. microti) but not againstM. bovisandM. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257–263, 2009,http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacyin vitroandex vivoagainst several species and strains ofMycobacteriumwith natural or acquired resistance to PZA, includingM. bovisandM. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activityper se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.

scholarly journals Characterization of DprE1-Mediated Benzothiazinone Resistance in Mycobacterium tuberculosis

2016 ◽  
Vol 60 (11) ◽  
pp. 6451-6459 ◽  
Author(s):  
Caroline Shi-Yan Foo ◽  
Benoit Lechartier ◽  
Gaëlle S. Kolly ◽  
Stefanie Boy-Röttger ◽  
João Neres ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Covalent Binding ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Lower Growth ◽  
Content Type ◽  
Covalent Inhibitors ◽  
Killing Action

ABSTRACTBenzothiazinones (BTZs) are a class of compounds found to be extremely potent against both drug-susceptible and drug-resistantMycobacterium tuberculosisstrains. The potency of BTZs is explained by their specificity for their target decaprenylphosphoryl-d-ribose oxidase (DprE1), in particular by covalent binding of the activated form of the compound to the critical cysteine 387 residue of the enzyme. To probe the role of C387, we used promiscuous site-directed mutagenesis to introduce other codons at this position intodprE1ofM. tuberculosis. The resultant viable BTZ-resistant mutants were characterizedin vitro,ex vivo, and biochemically to gain insight into the effects of these mutations on DprE1 function and onM. tuberculosis. Five different mutations (C387G, C387A, C387S, C387N, and C387T) conferred various levels of resistance to BTZ and exhibited different phenotypes. The C387G and C387N mutations resulted in a lower growth rate of the mycobacterium on solid medium, which could be attributed to the significant decrease in the catalytic efficiency of the DprE1 enzyme. All five mutations rendered the mycobacterium less cytotoxic to macrophages. Finally, differences in the potencies of covalent and noncovalent DprE1 inhibitors in the presence of C387 mutations were revealed by enzymatic assays. As expected from the mechanism of action, the covalent inhibitor PBTZ169 only partially inhibited the mutant DprE1 enzymes compared to the near-complete inhibition with a noncovalent DprE1 inhibitor, Ty38c. This study emphasizes the importance of the C387 residue for DprE1 activity and for the killing action of covalent inhibitors such as BTZs and other recently identified nitroaromatic inhibitors.

scholarly journals Developing Synergistic Drug Combinations To Restore Antibiotic Sensitivity in Drug-Resistant Mycobacterium tuberculosis

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Charles Omollo ◽  
Vinayak Singh ◽  
Elizabeth Kigondu ◽  
Antonina Wasuna ◽  
Pooja Agarwal ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Ex Vivo ◽  
Antibiotic Sensitivity ◽  
Infectious Agent ◽  
Drug Combinations ◽  
Major Facilitator Superfamily ◽  
Drug Resistant ◽  
Content Type

ABSTRACT Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis. We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S rRNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis. Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant. These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

scholarly journals Persistent Infection by a Mycobacterium tuberculosis Strain That Was Theorized To Have Advantageous Properties, as It Was Responsible for a Massive Outbreak

2015 ◽  
Vol 53 (11) ◽  
pp. 3423-3429 ◽  
Author(s):  
Laura Pérez-Lago ◽  
Yurena Navarro ◽  
Pedro Montilla ◽  
Iñaki Comas ◽  
Marta Herranz ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Virulent Strain ◽  
General Assumption ◽  
Host Population ◽  
Resistance Mutations ◽  
Single Nucleotide ◽  
Content Type ◽  
Treatment Interruptions ◽  
Highly Virulent

The strains involved in tuberculosis outbreaks are considered highly virulent and transmissible. We analyzed the case of a patient in Madrid, Spain, who was persistently infected over an 8-year period by the same BeijingMycobacterium tuberculosisstrain. The strain was responsible for a severe outbreak on Gran Canaria Island. The case provides us with a unique opportunity to challenge our assumptions aboutM. tuberculosisBeijing strains. No clinical/radiological findings consistent with a virulent strain were documented, and thein vitrogrowth rate of the strain in macrophages was only moderate. No secondary cases stemming from this prolonged active case were detected in the host population. The strain did not acquire resistance mutations, despite constant treatment interruptions, and it remained extremely stable, as demonstrated by the lack of single-nucleotide-polymorphism (SNP)-based differences between the sequential isolates. Our data suggest that the general assumption aboutM. tuberculosisBeijing strains having advantageous properties (in terms of virulence, transmissibility, and the tendency to acquire mutations and resistance) is not always accurate.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

2014 ◽  
Vol 80 (7) ◽  
pp. 2125-2132 ◽  
Author(s):  
Narjol Gonzalez-Escalona ◽  
Ruth Timme ◽  
Brian H. Raphael ◽  
Donald Zink ◽  
Shashi K. Sharma
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Clostridium Botulinum ◽  
Toxin Gene ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Group I

ABSTRACTClostridium botulinumis a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA+OrfX−) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA−OrfX+) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producingC. botulinumstrains: two strains with the HA+OrfX−cluster (69A and 32A) and one strain with the HA−OrfX+cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly availableC. botulinumgroup I strains revealed five distinct lineages. Strains 69A and 32A clustered with theC. botulinumtype A1 Hall group, and strain CDC297 clustered with theC. botulinumtype Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination ofC. botulinumgroup I strains and demonstrates the utility of this analysis in quickly differentiatingC. botulinumstrains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

scholarly journals Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Zhaojing Zong ◽  
Wei Jing ◽  
Jin Shi ◽  
Shu'an Wen ◽  
Tingting Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Novel Mutation ◽  
Multidrug Resistant ◽  
23S Rrna ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Significant Difference ◽  
Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

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