PCR amplification of <i>oprD</i> and IS<i>Pa</i>8 from carbapenem-resistant mutants of <i>P. aeruginosa</i>.

<p>(A) Primers that flanked the <i>oprD</i> gene were used to PCR-amplify the <i>oprD</i> gene giving an expected PCR product size is 1586 bp. Each lane is labeled with its respective DNA ladder (1 kb DNA ladder, Invitrogen), no template control (NTC), or <i>P. aeruginosa</i> isolate. (B) PCR amplification of IS<i>Pa</i>8 within the <i>oprD</i> gene in the nine carbapenem-resistant mutants. Primers ISPa8F1 and OprDRTR3 were used to map the approximate location of the IS<i>Pa</i>8 within the <i>oprD</i> gene. The smaller PCR products indicate IS<i>Pa</i>8 has inserted near the 3′ end of <i>oprD</i>, while larger PCR products indicate IS<i>Pa</i>8 is inserted near the 5′ end. No PCR product was observed for mutant 711M. (C) Primers OprDRTF2 and ISPa8R2 were used to confirm the location of IS<i>Pa</i>8 within the <i>oprD</i> gene in the nine isogenic mutants. Non-specific bands were amplified in PA42, mutant 711M, and mutant 922M suggesting that multiple IS<i>Pa</i>8 elements may be present within the genome. To confirm that IS<i>Pa</i>8 had inserted within the <i>oprD</i> gene or its flanking regions in mutants 711M and 922M, PCR products for these mutants shown in (A) were sequenced using primers OprDRTF2 or ISPa8F1.</p

Emergence of Carbapenem Resistance Due to the Novel Insertion Sequence IS<i>Pa</i>8 in <i>Pseudomonas aeruginosa</i>

<div><p>Chronic lung infections due to the persistence of <i>Pseudomonas aeruginosa</i> in cystic fibrosis patients are typically associated with the emergence of antibiotic resistance. The purpose of this study was to investigate the mechanisms responsible for the emergence of carbapenem resistance when a clinical isolate of <i>P. aeruginosa</i> collected from a patient with cystic fibrosis was challenged with meropenem. Nine carbapenem-resistant mutants were selected with subinhibitory concentrations of meropenem from a clinical isolate of <i>P. aeruginosa</i> and characterized for carbapenem resistance. Increased carbapenem MICs were associated with the identification of the novel insertion sequence IS<i>Pa</i>8 within <i>oprD</i> or its promoter region in all the mutants. The position of IS<i>Pa</i>8 was different for each of the mutants evaluated. In addition, Southern blot analyses identified multiple copies of IS<i>Pa</i>8 within the genomes of the mutants and their parent isolate. These data demonstrate that transposition of IS elements within the <i>Pseudomonas</i> genome can influence antibiotic susceptibility. Understanding the selective pressures associated with the emergence of antibiotic resistance is critical for the judicious use of antimicrobial chemotherapy and the successful treatment of bacterial infections.</p></div

Outer membrane analysis of <i>P. aeruginosa</i> PA42 and nine isogenic mutants.

<p>Outer membrane profiles were analyzed for the presence of the porin, OprD. PAO1 and a fully susceptible cystic fibrosis isolate, PA443, were included as positive controls. The locations of OprD and OprF proteins are indicated.</p

PCR amplification of the <i>oprF</i> gene in PAO1, PA42, and nine isogenic mutants.

<p>The expected PCR product size is 1,672(1 kb DNA ladder, Invitrogen), no template control (NTC), or <i>P. aeruginosa</i> isolates.</p

Pulsed Field Gel Electrophoresis and Southern blot analyses using IS<i>Pa</i>8-specific probe.

<p>(A) PFGE gel of <i>Spe</i>I chromosomal digests of wild-type strain PAO1, parent isolate PA42, and mutant 812M visualized using SYBR gold. (B) Southern blot of the gel depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091299#pone-0091299-g005" target="_blank">Figure 5(A)</a> using an IS<i>Pa</i>8-specific probe. Lane 1, PAO1 (negative control); lane 2, parent isolate PA42; lane 3, mutant 812M.</p