title: Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance.
abstract: 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 oprD transcription. The purpose of this study was to evaluate the molecular events responsible for decreased transcriptional expression of oprD in these strains. Expression of oprD 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 oprD 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 oprD. In summary, loss of OprD in all seven isolates was associated with mutations or insertions within oprD. 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 oprD gene by IS elements.
authors.affiliation: Department of Medical Microbiology and Immunology, Center for Research in Anti-Infectives and Biotechnology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wolter
authors.forename: Nancy D
authors.initials: ND
authors.lastname: Hanson
authors.forename: Philip D
authors.initials: PD
authors.lastname: Lister
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015780:Carbapenems / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D018272:Porins / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D015780:Carbapenems; D018272:Porins; C077157:OprD protein, Pseudomonas aeruginosa