Oligonucleotide primers used for PCR and real time RT-PCR.¹

<p>1 Restriction sites are indicated by underlined sequence</p><p>Oligonucleotide primers used for PCR and real time RT-PCR.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131534#t001fn001" target="_blank">¹</a></p

Bacterial strains or plasmids used in this study.

<p>Bacterial strains or plasmids used in this study.</p

Mutation, expression, and phenotypes of CmeR in selected <i>C</i>. <i>jejuni</i> isolates.

<p>*In relative to the expression level in NCTC 11168 as determined by RT-PCR</p><p>**Isolate is phenotypically classified as WEL</p><p>Mutation, expression, and phenotypes of CmeR in selected <i>C</i>. <i>jejuni</i> isolates.</p

Expression of CmeR in various isolates and its correlation with CmeABC expression.

<p>(A) Immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–9), and 11168Δ<i>cmeR</i> (lane 10) with the anti-CmeR antibody. The clinical isolates in lanes 2 to 9 are M63885, CT9:7, CB2:6, CB2:8, CB2:11, S13530, T37957A, and X7199, respectively. (B) Immunoblotting of whole cell proteins from 11168ΔcmeR (lane 1), CT2:2 (lane 2), and NCTC 11168 (lanes 3) with anti-CmeR, anti-CmeB, and anti-CmeA antibodies.</p

Effect of various mutations in CmeR and the promoter region on transcription of <i>cmeABC</i>.

<p>The names of the promoters used in the transcriptional fusions and β-galactosidase assays are indicated under each panel. Each promoter was assayed in the wild-type 81–176 background (A) and the 81–176ΔcmeR background (B). The data represent means with standard deviation from three independent experiments. The relative difference in transcription (fold change) due to repression by CmeR for each promoter is shown in (C) and was determined by comparison of transcription in the absence of CmeR (B) to the presence of CmeR (A). The unpaired Student’s t-test with Welch’s correction was used for comparison of the means with significance set at 0.05.</p

Emergence of ciprofloxacin-resistant mutants from WEL (circle) and OEL (triangle) isolates during treatment with ciprofloxacin.

<p>In panel A, the experiment was performed with an initial inoculum of 10⁷ CFU/ml of each isolate, while in panel B, the inoculum was 10⁶ CFU/mL for each isolate. The culture medium was MH broth containing 4 μg/mL of ciprofloxacin. Three WEL and OEL isolates were used in each experiment with cultures prepared in triplicate. Each point represents the number of ciprofloxacin-resistant mutants from a single culture. Bars represent mean log₁₀ CFU/mL for each group. Means for each phenotypic group were compared for each day with multiple unpaired Student’ t-tests and Holm-Šídák method for multiple comparisons. The significance level was set at 0.05.</p

Binding of CmeR to variants of the cmeABC promoter in different isolates.

<p>(A) Sequence alignment of the cmeABC promoter region illustrating the 16-base inverted repeat of the CmeR binding site shown in lowercase letters. The strain names are listed on the left of each sequence. All mutations differing from the 11168 promoter are highlighted in bold. (-) indicates a deleted base. (B) EMSA showing the binding of rCmeRSS to different promoter variants. The control probes include the NCTC 11168 probe (lanes 1–4) in panels I-III and the 81–176 probe (lanes 1–4) in panels IV to VI. The variant promoter probes include CT1:1 (panel I, lanes 5–8), CT1:9 (panel II, lanes 5–8), M32506 (panel III, lanes 5–8), X7199 (panel IV, lanes 5–8), CT3:7 (panel V, lanes 5–8), and CT9:20 (panel VI, lanes 5–8). For each probe, the amount of rCmeRSS used for the each reaction was 0 (lanes 1 and 5), 60 (lanes 2 and 6), 120 (lanes 3 and 7), and 180 ng (lanes 4 and 8), respectively. The rCmeRSS-DNA complexes are indicated with a “C” and the unbound promoter probe is indicated with a “P”.</p

Differential expression of CmeABC in clinical <i>Campylobacter jejuni</i> isolates.

<p>Expression was determined by immunoblotting of whole cell proteins from NCTC 11168 (lane 1), clinical isolates (lanes 2–11), and 11168Δ<i>cmeR</i> (lane 12) with anti-CmeB, anti-CmeC, anti-CmeA, and anti-major outer membrane protein (MOMP) antibodies. These broiler isolates in lanes 2 to 11 are CB1:6, CB1:14, CB 1:18, CB2:6, CB2:8, CB2:11, CB3:1, CB3:5, CB 3:14, and CB3:21. Isolates CB2:8, CB2:11, CB 3:1, CB3:5, CB3:14 and CB3:21 (lanes 6–11) were designated as having overexpression levels of CmeABC and isolates CB1:6, CB1:14, CB 1:18, and CB2:6 (lanes 2–5) as having wild-type levels of CmeABC. The major outer membrane protein (MOMP) was used as an internal control.</p

Inability of recombinant CmeR from isolate CT2:2 to bind to the promoter DNA of <i>cmeABC</i>.

<p>(A) Immunoblotting of purified rCmeRSS (lane 2; wild-type CmeR with C69 and C166 replaced with serine) and rCmeR-tr (lane 3; truncated CmeR after residue 193 from isolate CT2:2) with the anti-CmeR antibody. Lane 1 is the protein standard ladder. (B) EMSA showing binding of the 11168 cmeABC promoter by rCmeRSS (lanes 1–4) and rCmeR-tr (lanes 5–8). Proteins were added at 0, 60 (lanes 2 and 6), 120 (lanes 3 and 7), 180 ng (lanes 4 and 8). The locations of protein-DNA complexes and the probe are indicated.</p

Susceptibilities of <i>C. jejuni</i> 11168 and its efflux mutants to the selected plant extracts in the presence or absence of PAβN (20 µg/mL)^a, NMP (100 µg/mL) ^a, verapamil (100 µg/mL), reserpine (100 µg/mL) or CCCP (0.25µg/mL)^b.

<p>“<b>Fold diff.</b>” indicates fold difference, which is calculated using the formula: MIC without an EPI/MIC with an EPI. ≥4-fold changes are indicated in bold.</p>a<p>PAβN and NMP significantly (<i>p</i><0.05) reduced the MICs of the plant extracts in 11168, 11168F and 11168R, but not in 11168B (<i>p</i> > 0.05).</p>b<p>The effect of CCCP on the MICs of the plant extracts was only significant with 11168 (<i>p</i><0.05).</p