Open AccessInsertional inactivation of opr D in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance
Author(s) -
Wolter Daniel J,
Hanson Nancy D,
Lister Philip D
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2004.tb09639.x
Subject(s) - pseudomonas aeruginosa , biology , imipenem , insertion sequence , gene , efflux , microbiology and biotechnology , genetics , stop codon , pseudomonadaceae , bacteria , transposable element , genome
Recently, a Texas, USA hospital isolated seven Pseudomonas aeruginosa strains displaying dual resistance to fluoroquinolones and imipenem. These isolates were resistant to the fluoroquinolones through overexpression of the MexXY efflux pump and/or QRDR mutations and resistant to imipenem through downregulation of opr D transcription. The purpose of this study was to evaluate the molecular events responsible for decreased transcriptional expression of opr D in these strains. Expression of opr D could only be detected in two of the strains, but expression was very low as indicated by the high number of RT‐PCR cycles required to amplify the product. PCR was performed to amplify the opr D gene using primers upstream of the promoter and downstream of the structural gene. Amplified products were sequenced, and sequences were compared to wild‐type P. aeruginosa strain PAO1. Two isolates provided PCR products of the predicted size of 1586 bp, but sequencing revealed a single base change within the structural gene resulting in a premature stop codon. The other five isolates provided PCR products that were 1.3–1.6 kb larger than expected, suggesting the presence of large inserts. Sequence analysis indicated these inserts were novel insertion sequence elements transposed into different locations within opr D. In summary, loss of OprD in all seven isolates was associated with mutations or insertions within opr D. Although the point mutations that resulted in premature stop codons would explain the loss of the OprD protein in two isolates. This observation does not explain the observed decrease in transcriptional expression. This is the first report of carbapenem resistance occurring through insertional inactivation of the opr D gene by IS elements.