Rapid, Serial, Non-invasive Assessment of Drug Efficacy in Mice with Autoluminescent Mycobacterium ulcerans Infection

Tianyu Zhang,Jacques H Grosset,Paul J Converse,Eric L Nuermberger,Si-Yang Li,Jessica N Ricaldi

doi:10.1371/journal.pntd.0002598

Abstract

BackgroundBuruli ulcer (BU) caused by Mycobacterium ulcerans is the world's third most common mycobacterial infection. There is no vaccine against BU and surgery is needed for patients with large ulcers. Although recent experience indicates combination chemotherapy with streptomycin and rifampin improves cure rates, the utility of this regimen is limited by the 2-month duration of therapy, potential toxicity and required parenteral administration of streptomycin, and drug-drug interactions caused by rifampin. Discovery and development of drugs for BU is greatly hampered by the slow growth rate of M. ulcerans, requiring up to 3 months of incubation on solid media to produce colonies. Surrogate markers for evaluating antimicrobial activity in real-time which can be measured serially and non-invasively in infected footpads of live mice would accelerate pre-clinical evaluation of new drugs to treat BU. Previously, we developed bioluminescent M. ulcerans strains, demonstrating proof of concept for measuring luminescence as a surrogate marker for viable M. ulcerans in vitro and in vivo. However, the requirement of exogenous substrate limited the utility of such strains, especially for in vivo experiments.Methodology/Principal FindingFor this study, we engineered M. ulcerans strains that express the entire luxCDABE operon and therefore are autoluminescent due to endogenous substrate production. The selected reporter strain displayed a growth rate and virulence similar to the wild-type parent strain and enabled rapid, real-time monitoring of in vitro and in vivo drug activity, including serial, non-invasive assessments in live mice, producing results which correlated closely with colony-forming unit (CFU) counts for a panel of drugs with various mechanisms of action.Conclusions/SignificanceOur results indicate that autoluminescent reporter strains of M. ulcerans are exceptional tools for pre-clinical evaluation of new drugs to treat BU due to their potential to drastically reduce the time, effort, animals, compound, and costs required to evaluate drug activity.

Highlights

Buruli ulcer (BU) caused by Mycobacterium ulcerans is the world’s third most common mycobacterial disease with cases occurring on every continent, especially in certain humid tropical regions of the world [1,2]
Conclusions/Significance: Our results indicate that autoluminescent reporter strains of M. ulcerans are exceptional tools for pre-clinical evaluation of new drugs to treat BU due to their potential to drastically reduce the time, effort, animals, compound, and costs required to evaluate drug activity
The discovery and development of new drugs to improve the treatment of BU is greatly hampered by the slow growth rate of the organism, which requires up to 3 months to form countable colonies on solid media

Summary

Introduction

Buruli ulcer (BU) caused by Mycobacterium ulcerans is the world’s third most common mycobacterial disease with cases occurring on every continent, especially in certain humid tropical regions of the world [1,2]. Based on experiments in the mouse footpad model [6,7] and subsequent clinical studies [8,9,10,11,12], a regimen of streptomycin (STR) plus rifampin (RIF) for 2 months is recommended for treatment of BU [13], additional surgery including skin grafting may be necessary to repair large ulcers, contractures and deformities [10] Though this 2-month drug regimen does reduce numbers of colony-forming units (CFU), lesion size, and mycolactone levels [14,15], it has significant disadvantages, including STR’s requirement for parenteral administration and potential for oto- and vestibulotoxicity, while RIF causes challenging drug-drug interactions with many drugs, including anti-mycobacterial and anti-retroviral agents. The requirement of exogenous substrate limited the utility of such strains, especially for in vivo experiments

Methods

Results

Discussion

Conclusion