Bacterial viability of intracellular <i>C. jejuni</i>.

<p>Islands of polarized Caco-2 cells were infected with <i>C. jejuni</i> for 5 h in Hepes buffer, washed, incubated (3 h) with gentamicin (250 ìg/ml) in DMEM, washed again, and incubated for an additional 42 h in DMEM plus 10% FCS with a low dose of gentamicin (50 ìg/ml). At the indicated times, samples were prepared for bacterial viability assay. (A) Gentamicin killing assay showing the bacterial recovery of intracellular <i>C. jejuni</i> strains 108 containing pMA5-metK-luc (white and light grey bars) and 81–176 containing pMA5-metK-luc (dark grey and black bars) from Caco-2 cells at the indicated duration of infection. CFU were enumerated after 48 h of recovery on agar plates in a 0.2% oxygen (white and dark grey bars) and 5% oxygen (light grey and black bars) environment and indicated as CFU per well. (B) Bacterial viability as measured by bacterial luciferase reporter assay at the indicated time points. Values for results presented in (A) and (B) are the mean ± SEM of at 3 independent experiments in performed in duplicate.</p

Effect of ATP depletion on <i>C. jejuni</i> and <i>E. coli^inv</i> invasion.

<p>Islands of polarized Caco-2 cells were treated for 1 h with 3 mM of DNP and then infected with <i>C. jejuni</i> strain 108p4 (Red) and <i>E. coli^inv</i> (Green) for 2 h after which the cells were stained with WGA-Alexa fluor633 (Blue), fixed, and visualized with confocal microscopy. As a control, islands were treated with an equivalent amount of solvent acetone (final concentration: 0.3%) and infected. Note that DNP inhibits the invasion of <i>E. coli^inv</i> but not of <i>C. jejuni</i>.</p

Microtubule and actin cytoskeleton-independent <i>C. jejuni</i> invasion of Caco-2 cells.

<p>Islands of polarized Caco-2 cells were infected (2 h) with <i>C. jejuni</i> strain 108p4 (Red) in the absence of presence of the indicated actin cytoskeleton or microtubules disrupting or stabilizing drugs. Cells were fixed and stained with WGA-alexa fluor633 (Blue). Infected cells were visualized with confocal microscopy. The following drugs were used: (A) cytochalasin D (3 µM) and jasplakinolide (1 µM) added at 1 h prior to infection; (B) colchicine (10 µM) or paclitaxel (1 µM) added at 1 h prior to infection; (C) Colchicine (10 µM) added at 1 h after start of the infection and cells fixed at 2 h post infection. As control, cells were pre-treated with an equivalent amount of solvent DMSO (Final concentration 0.2%).</p

<i>C. jejuni</i> invades polarized Caco-2 islands via subvasion with high efficiency.

<p>Confocal laser microscopy on non-infected and <i>C. jejuni</i>-infected islands of polarized Caco-2 cells. (A) Uninfected island of Caco-2 cells stained with the membrane marker WGA-Alexa fluor633 (Blue) and an anti-occludin antibody (Green) showing the presence of tight junctions. (B) Caco-2 cells (Blue) at 1 h of infection in DMEM showing <i>C. jejuni</i> strain 108p4 (Red) mostly located at the basal side of cells near the edge of the island of polarized cells. (C) Caco-2 cells (Blue) at 5 h of infection in DMEM demonstrating intracellular <i>C. jejuni</i> strain 108p4 (Red) at the center of the island of cells with tight junctions (Green). (D). Polarized Caco-2 cells (Blue) infected (1 h and 5 h) with a mixture of <i>C. jejuni</i> strains 108p4 (Red) and 81–176 (Green) showing invasion of Caco-2 cells by both strains. Transversal optical sections of the cells are depicted at the bottom of each panel to show the location of the bacteria relative to the cell basis.</p

The Natural Antimicrobial Carvacrol Inhibits <i>Campylobacter jejuni</i> Motility and Infection of Epithelial Cells

<div><p>Background</p><p>Natural compounds with anti-microbial properties are attractive reagents to reduce the use of conventional antibiotics. Carvacrol, the main constituent of oregano oil, inhibits the growth of a variety of bacterial foodborne pathogens. As concentrations of carvacrol may vary <i>in vivo</i> or when used in animal feed, we here investigated the effect of subinhibitory concentrations of the compound on major virulence traits of the principal bacterial foodborne pathogen <i>Campylobacter jejuni</i>.</p><p>Methods/Principal Findings</p><p>Motility assays revealed that subinhibitory concentrations of carvacrol inhibited the motility of <i>C. jejuni</i> without affecting bacterial growth. Immunoblotting and electron microscopy showed that carvacrol-treated <i>C. jejuni</i> still expressed flagella. The loss of motility was not caused by reduced intracellular ATP levels. <i>In vitro</i> infection assays demonstrated that subinhibitory concentrations of carvacrol also abolished <i>C. jejuni</i> invasion of human epithelial cells. Bacterial uptake of invasive <i>Escherichia coli</i> was not blocked by carvacrol. Exposure of <i>C. jejuni</i> to carvacrol prior to infection also inhibited cellular infection, indicating that the inhibition of invasion was likely caused by an effect on the bacteria rather than inhibition of epithelial cell function.</p><p>Conclusions/Significance</p><p>Bacterial motility and invasion of eukaryotic cells are considered key steps in <i>C. jejuni</i> infection. Our results indicate that subinhibitory concentrations of carvacrol effectively block these virulence traits by interfering with flagella function without disturbing intracellular ATP levels. These results broaden the spectrum of anti-microbial activity of carvacrol and support the potential of the compound for use in novel infection prevention strategies.</p></div

Effect of carvacrol on <i>C. jejuni</i> growth.

<p>(A) Growth of <i>C. jejuni</i> strains 108 and 81116 in HI supplemented with increasing concentrations of carvacrol as determined after 16 h of incubation. The maximum sub-inhibitory concentration (SIC) is indicated by an arrow. (B) Growth kinetics of <i>C. jejuni</i> strain 108 with or without carvacrol at SIC monitored during a 24 h-period. Results are mean values ± standard error of three independent experiments.</p

Partial restoration of invasion in carvacrol-treated <i>C. jejuni</i>.

<p>Invasion of <i>C. jejuni</i> (strain 108) into INT-407 cells was determined in the absence (filled bars) and presence (open bars) of 0.2 mM of carvacrol, without (−) and with (+) centrifugation of the bacteria onto the cells at the start of the infection. Note the five-fold increase in invasion by <i>C. jejuni</i> grown with carvacrol after centrifugation. Results are expressed as percentage of invasion in the absence of carvacrol and are mean values ± standard error of at least three independent experiments. *** <i>p</i>≤0.001.</p

Inhibition of <i>C. jejuni</i> motility by carvacrol.

<p>Swarming of <i>C. jejuni</i> strain 108 (A) and 81116 (B) and the non-motile mutants 108Δ<i>motAB</i> (C) and in 81116Δ<i>motAB</i> (D) in thioglycollate agar in the absence and presence of the indicated concentrations of carvacrol after 24 h of growth. The carvacrol solvent ethanol (EtOH) served as negative control.</p

Lysozyme MIC levels of strain 10 after passaging.

<p>Lysozyme MIC of strain 10 passaged four times onto Colombia agar plates containing two-fold increasing concentrations of lysozyme (start concentration: 62.5 µg/ml). In general, within 4 passages the lysozyme MIC increased towards levels observed in natural lysozyme resistant strains (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036281#pone-0036281-g001" target="_blank">Figure 1</a>). * Lysozyme MIC level after sub-culturing lysozyme resistant strains in the absence of lysozyme. Values represent the MIC levels of an example of a selection procedure.</p

Effect of carvacrol on bacterial invasion of INT-407 cells.

<p>Invasion of INT-407 cells by <i>C. jejuni</i> (strain 108) and <i>E. coli inv</i> in the absence (filled bars) and presence (open bars) of 0.2 mM of carvacrol. Bacteria used for inoculation were grown in HI broth without (−) or with (+) carvacrol. Invasion was determined using the gentamicin protection assay. Note that the reduction in invasion was specific for <i>C. jejuni</i> and most effective when carvacrol was present before and during the infection assay. Results are expressed as percentage of invasion in the absence of carvacrol and are mean values ± standard error from at least three independent experiments. *** <i>p</i>≤0.001; n.s. not statistically significant.</p