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

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01948-20 ◽

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.

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

Infection and Immunity ◽

10.1128/iai.03080-14 ◽

2015 ◽

Vol 83 (6) ◽

pp. 2213-2223 ◽

Cited By ~ 1

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.

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Efflux Attenuates the Antibacterial Activity of Q203 in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02637-16 ◽

2017 ◽

Vol 61 (7) ◽

Cited By ~ 13

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.

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Vitamin C Potentiates the Killing of Mycobacterium tuberculosis by the First-Line Tuberculosis Drugs Isoniazid and Rifampin in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02165-17 ◽

2018 ◽

Vol 62 (3) ◽

Cited By ~ 9

Author(s):

Catherine Vilchèze ◽

John Kim ◽

William R. Jacobs

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Vitamin C ◽

Mouse Serum ◽

First Line ◽

Content Type ◽

Tb Treatment ◽

High Concentrations

ABSTRACT The treatment of drug-susceptible tuberculosis (TB) is long and cumbersome. Mismanagement of TB treatment can lead to the emergence of drug resistance in patients, so shortening the treatment duration could significantly improve TB chemotherapy and prevent the development of drug resistance. We previously discovered that high concentrations of vitamin C sterilize cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis . Here, we tested subinhibitory concentration of vitamin C in combination with TB drugs against M. tuberculosis in vitro and in a mouse model of M. tuberculosis infection. In vivo , we showed that the vitamin C level in mouse serum can be increased by intraperitoneal injection of vitamin C to reach vitamin C levels close to the concentrations required for activity in vitro . Although vitamin C had no activity by itself in M. tuberculosis -infected mice, the combination of vitamin C with the first-line TB drugs isoniazid and rifampin reduced the bacterial burden in the lungs of M. tuberculosis -infected mice faster than isoniazid and rifampin combined in two independent experiments. These experiments suggest that the addition of vitamin C to first-line TB drugs could shorten TB treatment. Vitamin C, an inexpensive and nontoxic compound, could easily be added to the TB pharmacopeia to substantially improve chemotherapy outcome, which would have a significant impact on the worldwide TB community.

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

mBio ◽

10.1128/mbio.02645-20 ◽

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.

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Characterization of the Retrocyclin Analogue RC-101 as a Preventative of Staphylococcus aureus Nasal Colonization

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00619-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5338-5346 ◽

Cited By ~ 12

Author(s):

Ryan P. Lamers ◽

Colleen R. Eade ◽

Alan J. Waring ◽

Amy L. Cole ◽

Alexander M. Cole

Keyword(s):

Staphylococcus Aureus ◽

Ex Vivo ◽

Treatment Options ◽

Nasal Colonization ◽

Proinflammatory Response ◽

Content Type ◽

Bactericidal Effects ◽

Prevention Of Nosocomial Infection

ABSTRACTNasal colonization ofStaphylococcus aureusis a risk factor for pathogenic autoinfection, particularly in postoperative patients and the immunocompromised. As such, standardized preoperative nasal decolonization ofS. aureushas become a major consideration for the prevention of nosocomial infection. However, only a few treatment options for nasal decolonization are currently available, with resistance to these approaches already a concern. Here we have identified the macrocyclic θ-defensin analogue RC-101 as a promising anti-S. aureusagent for nasal decolonization. RC-101 exhibits bactericidal effects againstS. aureuswith the use ofin vitroepithelium-free systems, while also preventing the pathogen's proliferation and attachment in anex vivohuman nasal epithelial cell adhesion model and an organotypic model of human airway epithelia. Peptide concentrations as low as 2.5 μM elicited significant reductions inS. aureusgrowth in epithelium-free systems, with 10 μM concentrations being completely bactericidal for all strains tested, including USA300. Inex vivonasal colonization models, RC-101 significantly reduced adherence, survival, and proliferation ofS. aureuson human nasal epithelia. Reductions inS. aureusviability were evident in these assays, with as little as 1 μg of peptide per tissue, while 10 μg of RC-101 completely prevented adhesion of all strains tested. Furthermore, RC-101 did not exhibit cellular toxicity to human nasal epithelia at concentrations up to 200 μM, nor did it induce a proinflammatory response in these cells. Collectively, the findings of this study identify RC-101 as a potential preventative ofS. aureusnasal colonization.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00936-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7693-7699 ◽

Cited By ~ 7

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.

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Characterization of DprE1-Mediated Benzothiazinone Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01523-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6451-6459 ◽

Cited By ~ 19

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02554-20 ◽

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.

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Bioluminescence for Assessing Drug Potency against Nonreplicating Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00528-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4012-4019 ◽

Cited By ~ 23

Author(s):

Anthony Vocat ◽

Ruben C. Hartkoorn ◽

Benoit Lechartier ◽

Ming Zhang ◽

Neeraj Dhar ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Time Course ◽

Drug Efficacy ◽

Luciferase Assay ◽

Luciferase Expression ◽

Content Type ◽

Drug Potency ◽

Bactericidal Effects

ABSTRACTTargeting dormantMycobacterium tuberculosisrepresents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependentM. tuberculosis18b strain as a tool for assessing drug potency against nonreplicating bacteria bothin vitroandin vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing theluxCDABEoperon fromPhotorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacyin vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications.

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