Molecular Diagnosis of Fluoroquinolone Resistance in Mycobacterium tuberculosis

As a consequence of the use of fluoroquinolones (FQ), resistance to FQ has emerged, leading to cases of nearly untreatable and extensively drug-resistant tuberculosis. Mutations in DNA gyrase represent the main mechanism of FQ resistance. A full understanding of the pattern of mutations found in FQ-resistant (FQ^r) clinical isolates, and of their proportions, is crucial for improving molecular methods for the detection of FQ resistance in Mycobacterium tuberculosis. In this study, we reviewed the detection of FQ resistance in isolates addressed to the French National Reference Center for Mycobacteria from 2007 to 2012, with the aim of evaluating the performance of PCR sequencing in a real-life context. gyrA and gyrB sequencing, performed prospectively on M. tuberculosis clinical isolates, was compared for FQ susceptibility to 2 mg/liter ofloxacin by the reference proportion method. A total of 605 isolates, of which 50% were multidrug resistant, were analyzed. The increase in FQ^r strains among multidrug-resistant (MDR) strains during the time of the study was alarming (8% to 30%). The majority (78%) of the isolates with gyrA mutations were FQ^r, whereas only 36% of those with gyrB mutations were FQ^r. Only 12% of the FQ^r isolates had a single mutation in gyrB. Combined gyrA and gyrB sequencing led to >93% sensitivity for detecting resistance. The analysis of the four false-positive and the five false-negative results of gyrA and gyrB sequencing illustrated the actual limitations of the reference proportion method. Our data emphasize the need for combined gyrA and gyrB sequencing in the investigation of FQ susceptibility in M. tuberculosis and challenge the validity of the current phenotype-based approach as the diagnostic gold standard for determining FQ resistance.     

Intestinal over-expression of iron transporters induces iron overload in birds in captivity

Hereditary hemochromatosis (HH) is a frequent genetic disease of older subjects of northern European descent. It is characterized by increased iron absorption and severe iron overloading in parenchymal organs. A similar disturbance of iron metabolism occurs in specific animal species in captivity. To address the key features leading to high absorption and thus to iron overload in these animals, we have studied the two iron transport proteins DMT1 and Ireg1 in the best-known susceptible species, the mynah bird. Here, we show that these birds have a high expression of DMT1 in the duodenum and also a strikingly high expression of Ireg1 along the whole small intestine. We believe that the iron accumulation in susceptible species only occurs in captivity because of a genotypic adaptation to their natural environment, where contrary to captivity, dietary iron is hardly available. The Caucasian population carrying mutations leading to iron overload today may have also benefited from the genetic advantage of up-regulating iron transport millennia ago, when dietary iron was scarce.