Preclinical efforts to discover and develop new drugs and vaccines for tuberculosis are hampered by the reliance on colony-forming unit (CFU) counts as primary outcomes for in vivo efficacy studies ...and the slow growth of Mycobacterium tuberculosis. The utility of bioluminescent M. tuberculosis reporter strains for real-time in vitro and ex vivo assessment of drug and vaccine activity has been demonstrated but a simple, non-invasive, real-time surrogate marker to replace CFU counts for real-time evaluation of drug and vaccine efficacy in vivo has not been described. We describe the development of a fully virulent and stable autoluminescent strain of M. tuberculosis and proof-of-concept experiments demonstrating its utility for in vivo bioluminescence imaging to assess the efficacy of new drugs and vaccines for tuberculosis in a mouse model. Relative light unit (RLU) counts paralleled CFU counts during the active phase of bacterial growth, with a lower limit of detection of approximately 10(6) CFU in live, anesthetized mice. Experiments distinguishing active from inactive anti-tuberculosis drugs and bacteriostatic drug effects from bactericidal effects were completed in less than 5 days. The ability of a recombinant BCG vaccine to limit bacterial growth was demonstrated within 3 weeks. Use of this autoluminescent reporter strain has the potential to drastically reduce the time, effort, animals and costs consumed in the evaluation of drug activity in vitro and the in vivo assessment of drug and vaccine efficacy.
Mycobacterium abscessus is an emerging opportunistic human pathogen that naturally resists most major classes of antibiotics, making infections difficult to treat. Thus far, little is known about
...physiology, pathogenesis, and drug resistance. Genome-wide analyses have comprehensively catalogued genes with essential functions in Mycobacterium tuberculosis and Mycobacterium avium subsp.
(here,
) but not in
. By optimizing transduction conditions, we achieved full saturation of TA insertion sites with Himar1 transposon mutagenesis in the
ATCC 19977
genome, as confirmed by deep sequencing prior to essentiality analyses of annotated genes and other genomic features. The overall densities of inserted TA sites (85.7%), unoccupied TA sites (14.3%), and nonpermissive TA sites (8.1%) were similar to results in
and
. Of the 4,920 annotated genes, 326 were identified as essential, 269 (83%) of which have mutual homology with essential
genes, while 39 (12%) are homologous to genes that are not essential in
and
, and 11 (3.4%) only have homologs in
. Interestingly, 7 (2.1%) essential
genes have no homologs in either
or
, two of which were found in phage-like elements. Most essential genes are involved in DNA replication, RNA transcription and translation, and posttranslational events to synthesize important macromolecules. Some essential genes may be involved in
pathogenesis and antibiotics response, including certain essential tRNAs and new short open reading frames. Our findings will help to pave the way for better understanding of
and benefit development of novel bactericidal drugs against
.
Limited knowledge regarding
pathogenesis and intrinsic resistance to most classes of antibiotics is a major obstacle to developing more effective strategies to prevent and mitigate disease. Using optimized procedures for Himar1 transposon mutagenesis and deep sequencing, we performed a comprehensive analysis to identify
genetic elements essential for
growth and compare them to similar data sets for
and
subsp.
. Most essential
genes have mutual homology with essential
genes, providing a foundation for leveraging available knowledge from
to develop more effective drugs and other interventions against
. A small number of essential genes unique to
deserve further attention to gain insights into what makes
different from other mycobacteria. The essential genes and other genomic features such as short open reading frames and noncoding RNA identified here will provide useful information for future study of
pathogenicity and new drug development.
A regimen comprised of bedaquiline (BDQ, or B), pretomanid, and linezolid (BPaL) is the first oral 6-month regimen approved by the U.S. Food and Drug Administration and recommended by the World ...Health Organization for the treatment of extensively drug-resistant tuberculosis. We used a well-established BALB/c mouse model of tuberculosis to evaluate the treatment-shortening potential of replacing bedaquiline with either of two new, more potent diarylquinolines, TBAJ-587 and TBAJ-876, in early clinical trials. We also evaluated the effect of replacing linezolid with a new oxazolidinone, TBI-223, exhibiting a larger safety margin with respect to mitochondrial toxicity in preclinical studies. Replacing bedaquiline with TBAJ-587 at the same 25-mg/kg dose significantly reduced the proportion of mice relapsing after 2 months of treatment, while replacing linezolid with TBI-223 at the same 100-mg/kg dose did not significantly change the proportion of mice relapsing. Replacing linezolid or TBI-223 with sutezolid in combination with TBAJ-587 and pretomanid significantly reduced the proportion of mice relapsing. In combination with pretomanid and TBI-223, TBAJ-876 at 6.25 mg/kg was equipotent to TBAJ-587 at 25 mg/kg. We conclude that replacement of bedaquiline with these more efficacious and potentially safer diarylquinolines and replacement of linezolid with potentially safer and at least as efficacious oxazolidinones in the clinically successful BPaL regimen may lead to superior regimens capable of treating both drug-susceptible and drug-resistant TB more effectively and safely.
Community-acquired meticillin-resistant
Staphylococcus aureus (MRSA) is becoming an important public-health problem. New strains of
S aureus displaying unique combinations of virulence factors and ...resistance traits have been associated with high morbidity and mortality in the community. Outbreaks of epidemic furunculosis and cases of severe invasive pulmonary infections in young, otherwise healthy people have been particularly noteworthy. We review the characteristics of these new strains of community-acquired MRSA that have contributed to their pathogenicity and discuss new approaches to the diagnosis and management of suspected and confirmed community-acquired MRSA infections.
Tigecycline is used in multidrug regimens for salvage therapy of
infections but is often poorly tolerated and has no oral formulation. Here, we report similar
activity of two newly approved ...tetracycline analogs, omadacycline and eravacycline, against 28 drug-resistant clinical isolates of
complex. Since omadacycline and eravacycline appear to be better tolerated than tigecycline and since omadacycline is also formulated for oral dosing, these tetracycline analogs may represent new treatment options for
infections.
Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and ...manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.
Background. Preclinical evaluation of tuberculosis drugs is generally limited to mice. However, necrosis and hypoxia, key features of human tuberculosis lesions, are lacking in conventional mouse ...strains. Methods. We used C3HeB/FeJ mice, which develop necrotic lesions in response to Mycobacterium tuberculosis infection. Positron emission tomography in live infected animals, postmortem pimonidazole immunohistochemistry, and bacterial gene expression analyses were used to assess whether tuberculosis lesions in C3HeB/FeJ are hypoxic. Efficacy of combination drug treatment, including PA-824, active against M. tuberculosis under hypoxic conditions, was also evaluated. Results. Tuberculosis lesions in C3HeB/FeJ (but not BALB/c) were found to be hypoxic and associated with up-regulation of known hypoxia-associated bacterial genes (P<.001). Contrary to sustained activity reported elsewhere in BALB/c mice, moxifloxacin and pyrazinamide (MZ) combination was not bactericidal beyond 3 weeks in C3HeB/FeJ. Although PA-824 added significant activity, the novel combination of PA-824 and MZ was less effective than the standard first-line regimen in C3HeB/FeJ. Conclusions.We demonstrate that tuberculosis lesions C3HeB/FeJ are hypoxic. Activities of some key tuberculosis drug regimens in development are represented differently in C3HeB/FeJ versus BALB/c mice. Because /FeJ display key features of human tuberculosis, this strain warrants evaluation as a more pathologically relevant model for preclinical studies.
This is a review of the preclinical efficacy testing of new antituberculosis drug candidates. It describes existing dynamic
and
models of antituberculosis chemotherapy and their utility in ...preclinical evaluations of promising new drugs and combination regimens, with an effort to highlight recent developments. Emphasis is given to the integration of quantitative pharmacokinetic/pharmacodynamic analyses and the impact of lesion pathology on drug efficacy. Discussion also includes
models of chemotherapy of latent tuberculosis infection.
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