Sequencing of the rpoB Gene in Legionella pneumophila and Characterization of Mutations Associated with Rifampin Resistance in the Legionellaceae

Abstract

ABSTRACT Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis , and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae . Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis , was sequenced from 18 rifampin-resistant Legionella isolates representing four species ( L. bozemanii , L. longbeachae , L. micdadei , and L. pneumophila ), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae .