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Intracellular penetration capacity of Mycobacterium tuberculosis drug-resistant isolates using a murine macrophage model

Abstract number: P1964

Rey E., Tudó G., Borrell S., Alcaide F., Codina G., Coll P., Martín N., Mick V., Montemayor M., Salvadó M., Vicente E., González J.

Objectives: To determine the penetration capacity into macrophages of Mycobacterium tuberculosis isolates resistant to different drugs compared with susceptible clinical isolates.

Methods:Isolates: Seventeen susceptible isolates were collected from tuberculosis (TB) patients of the Hospital Clinic of Barcelona. Forty-two isolates resistant to isoniazid (H) and/or rifampicin (R) were selected from an isolate collection of TB patients attended in five university hospitals of the Barcelona area according to their molecular mechanism associated with resistance. Of these, 5 isolates showed mutation in the katG gene, 11 mutation in the inhA gene, 5 mutation in the rpoB gene, 11 mutations in the katG gene and rpoB and 10 were wild-type at these positions. Mutations in the katG gene were at position 315.

Measurement of M. tuberculosis penetration in macrophages: Murine macrophages from the cell line J774.2 were used as an in vitro infection model. The cell monolayer was infected with M. tuberculosis isolates in duplicate using a 1:1 MOI. After 3 hours of incubation, the monolayers were washed to remove extracellular bacteria. The monolayers were then lysed. Cellular lysates and inoculum were plated on Middlebrook 7H11 plates and incubated at 37°C, being read at the end of 3 and 4 weeks. Penetration (%) was calculated as: total CFU cellular lysates / total CFU inoculum.

Results: Lower penetration percentages were observed in resistant isolates inside the macrophages compared with susceptible isolates (p < 0.05). H-resistant isolates with mutation in the katG gene and multidrug-resistant (MDR) isolates with mutation in the rpoB and katG genes had a lower penetration percentage than isolates with other mutations or susceptible isolates (p < 0.05). Resistant isolates with mutations in the inhA or rpoB genes or resistant without detected mutations had a similar behaviour to that of susceptible isolates. No significant difference was found between isolates with mutation in the katG gene and isolates with mutations in the katG and rpoB genes.

Conclusions: H-monoresistant and MDR isolates of M. tuberculosis with mutation at codon 315 in the katG gene have a lower penetration capacity inside murine macrophages, which could be associated with lower virulence. The lack of a significant difference between isolates with a katG mutation and with katG and rpoB mutations suggests the absence of an accumulative effect.

Session Details

Date: 10/04/2010
Time: 00:00-00:00
Session name: Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Vienna, Austria, 10 - 13 April 2010
Presentation type:
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