Carbapenem susceptibility and gene expression data for <i>P. aeruginosa</i> isolates.

a<p>WT, wild-type isolate; CF, clinical isolate from cystic fibrosis patient; P, parent isolate; M, mutant of PA42.</p>b<p>Subinhibitory concentration (µg/mL) of meropenem used for mutant selection.</p>c<p>IPM, imipenem; MEM, meropenem; DOR, doripenem.</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

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

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

Apical exosomes released from <i>C. parvum</i>-infected biliary epithelium display anti-<i>C. parvum</i> activity.

<p>(A) Effects of incubation with isolated exosomes from the biliary epithelium on <i>C. parvum</i> viability. Exosomes were isolated from the basolateral or apical supernatants of H69 monolayers of non-infected control, cells of <i>C. parvum</i> infection for 24 h, and cells stably expressing TLR4-DN after infection for 24 h. Freshly excysted <i>C. parvum</i> sporozoites were incubated with isolated exosomes (as indicated) for 2 h, and parasite viability was assessed by propidium iodide staining. (B) and (C) Effects of incubation with isolated apical exosomes from the biliary epithelium on the infectivity of <i>C. parvum</i> sporozoites. The same number of freshly excysted <i>C. parvum</i> sporozoites was incubated with exosomes isolated from H69 or 603B monolayers for 2 h and then added to H69 cells for 2 h. Parasite infection burden was measured by real-time PCR (B) and fluorescence microscopy (C). Pre-incubation of isolated exosomes abolished their inhibitory effects on <i>C. parvum</i> infectivity. <i>C. parvum</i> parasites were stained in green using a specific antibody and cell nuclei in blue by DAPI. Scale bars = 5 µm; *, p<0.05 ANOVA versus medium control.</p

Downregulation of <i>let-7</i> miRNAs is associated with <i>C. parvum</i>-induced exosome release from epithelial cells.

<p>(A) Functional manipulation of <i>let-7</i>/miR-98 causes reciprocal alterations in SNAP23 expression at the mRNA and protein levels. H69 cells were treated with various doses of <i>let-7i</i> precursor or anti-<i>let-7i</i> for 24 h, followed by Western blot for SNAP23 protein (upper panel) and real-time PCR of SNAP23 mRNA (lower panel). Whereas a significant decrease or increase in SNAP23 protein content was detected in cells treated with <i>let-7i</i> precursor or anti-<i>let-7i</i>, respectively, no changes in SNAP23 mRNA levels were observed in the treated cells. (B) Anti-<i>let-7i</i> shows no effect on the phosphorylation of SNAP23 in H69 cells. Cells were treated with anti-<i>let-7i</i> for 24 h, followed by IP for phosphorylated SNAP23. (C) Overexpression of <i>let-7i</i> blocks <i>C. parvum</i>-induced upregulation of SNAP23. H69 cells were transfected with <i>let-7i</i> precursor for 48 h and then exposed to <i>C. parvum</i> infection for 24 h. Expression of SNAP23 was measured by real-time PCR and Western blot. (D) Functional manipulation of <i>let-7i</i> causes alterations in <i>C. parvum</i>-induced release of apical exosomes. H69 monolayers were treated with a non-specific anti-miR control or anti-<i>let-7i</i> or <i>let-7i</i> precursor, followed by exposure to <i>C. parvum</i> infection for 24 h. Exosomes released into the apical supernatants were isolated and quantified. Data are averages of three independent experiments. *, p<0.05 ANOVA versus the non-infected control; ^#, p<0.05 ANOVA versus the control anti-miR.</p

The <i>let-7</i> miRNAs target SNAP23 and are downregulated in epithelial cells following <i>C. parvum</i> infection.

<p>(A) <i>C. parvum</i> infection downregulates expression of the <i>let-7</i> miRNA family. H69 cells were exposed to <i>C. parvum</i> infection for 12 h, followed by miRNA analysis using mercury LNA array, Northern blot, and real-time PCR. A heat map of the <i>let-7</i> miRNA family is shown and expression levels are presented as the log₂ (Hy⁵/Hy³) ratios. Representative Northern blot for <i>let-7</i>/miR-98 in H69 and 603B cells and real-time PCR quantification of miR-98 in H69 cells are shown. (B) The schematic of SNAP23 mRNA showed a potential binding site in its 3′UTR for the <i>let-7</i> miRNA family in humans and in mice. The SNAP23 3′UTR sequence covering the potential binding sites for the <i>let-7</i> miRNA family was inserted into the pMIR-REPORT luciferase plasmid. A control plasmid with the mutant 3′UTR sequence was also generated for control. (C) Binding of <i>let-7</i>/miR-98 miRNAs to the potential binding site in the SNAP23 3′UTR results in translational suppression. Cells were transfected with the pMIR-REPORTER luciferase constructs and treated with anti-miRs or precursors to miR-98, or non-specific oligo control, for 24 h, followed by luciferase analysis. *, p<0.05 ANOVA versus the non-infected control (in A) or empty vector control (in C); ^#, p<0.05 ANOVA versus the control precursor or control anti-miR (in C).</p

TLR4-dependent luminal release of exosomes during <i>C. parvum</i> biliary infection in mice.

<p>(A) and (B) Luminal release of biliary exosomes during biliary cryptosporidiosis in the wild type and TLR4-deficient mice. <i>C. parvum</i> oocysts were injected into the gallbladder of wild-type or TLR4-deficient mice, and liver tissues were collected one week post-injection. Quantitative analysis detected a higher infection burden and a lower amount of luminal exosome content in TLR4-deficent mice. (C) Abundant exosome-like microvesicles were observed in the luminal region of the wild-type mice one week post-infection, compared with that in the TLR4-deficent mice by transmission EM (b and d are higher magnifications in a and c, respectively). Scale bars = 1 µm; * p<0.05 ANOVA versus the wild-type.</p