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1 Department of Pathology, Bacteriology and Avian Diseases, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
2 Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Unit, Brusselsesteenweg 370, 9090 Melle, Belgium
3 Department of Virology, Parasitology and Immunology, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
4 Department of Reproduction, Obstetrics and Herd Health, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Correspondence
Kim Van Deun
kim.vandeun{at}ugent.be
Received 16 April 2007
Accepted 25 June 2007
Abbreviations: CDT, cytolethal distending toxin; IL-8, interleukin-8; TNF, tumour necrosis factor.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Several virulence factors are considered to be important for the induction of gastroenteritis, such as resistance to bile salts (Lin et al., 2003), invasion of epithelial cells (Russell et al., 1993) and cytolethal distending toxin (CDT) production (Konkel et al., 2001). Cell invasion could result in cellular injury, leading to reduced absorptive capacity of the intestine, whereas CDT production is important for interleukin-8 (IL-8) release by intestinal cells in vitro (Hickey et al., 1999, 2000) and thus plays an important role in the host mucosal inflammatory response.
Although poultry meat is considered an important source of C. jejuni infections for humans, it is not known whether all poultry-associated C. jejuni strains are capable of causing disease in humans. We therefore compared the capability for epithelial cell invasion, CDT production, bile-salt resistance and IL-8 induction of poultry and human isolates.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Bacterial strains. In this study, 20 C. jejuni strains of poultry origin and 24 clinical isolates (kindly provided by Professor Peter Vandamme, Ghent University) were used. The choice of strains was based on their genotypic heterogeneity, which was determined by PFGE with KpnI and SmaI (Amersham Biosciences) according to the PulseNet USA protocol (http://www.cdc.gov/pulsenet/protocols/campy_protocol.pdf). PFGE profiles were clustered with BioNumerics software (version 4.0, Applied Maths) using the Dice coefficient and the unweighted pair group method using arithmetic averages (UPGMA) clustering algorithm. PFGE clusters were defined on a similarity level of 80 %. All strains were routinely cultured in Nutrient Broth No. 2 (Oxoid), supplemented with Campylobacter-specific growth supplements (SR117 and SR0232, Oxoid) and 5 % lysed horse blood (E&O Laboratories) at 42 °C under microaerobic conditions (5 % O2, 5 % CO2, 5 % H2, 85 % N2). For certain experiments, horse blood was omitted. Care was taken to pass the strains no more than five times in vitro.
Before use in virulence assays, all C. jejuni strains were passed through day-old specific pathogen-free (SPF) White Leghorn Chickens. Chicks were orally inoculated with 2x107 organisms in 0.2 ml PBS. Controls were sham-inoculated with PBS alone. At 24 h post-inoculation, cloacal swabs were streaked on modified charcoal cefoperazone deoxycholate agar plates (mCCDA; CM0739, Oxoid) containing Campylobacter-specific growth supplements (SR0155 and SR0232, Oxoid) for 48 h at 42 °C under microaerobic conditions. Bacteria were collected by washing plates with PBS, and the aliquoted suspension was stored in horse blood at –80 °C.
For the CDT assay, bacterial cell lysates were prepared by sonication. Bacterial cultures were grown in 40 ml selective broth with 5 % lysed horse blood for 19 h at 42 °C in a microaerobic atmosphere to a density of 5x107 c.f.u. ml–1 and harvested by centrifugation at 900 g for 20 min at 4 °C. The bacteria were washed once with 3 ml PBS and resuspended in 3 ml PBS. The pellet was sonicated on ice by eight 30 s pulses with an XL 2015 Sonicator (Misonix) followed by centrifugation at 6000 g for 10 min at 4 °C. The supernatant was filter-sterilized by subsequent passage through 0.45 and 0.20 µm pore-size filters (IWAKI, International Medical) and stored at –80 °C.
Cell lines. The HEp-2 cell line was obtained from the European Collection of Cell Cultures (ECACC). Cells were grown in Eagle's Minimum Essential Medium (EMEM; Gibco, Invitrogen) supplemented with 10 % fetal calf serum, 1 % glutamine, 1 % non-essential amino acids and, unless stated otherwise, 100 U penicillin ml–1 and 0.1 mg streptomycin ml–1 (Gibco) at 37 °C in a 5 % CO2 atmosphere.
The human colon carcinoma cell line T84 (ECACC) was grown in 44 % Dulbecco's Minimal Essential Medium (DMEM), 44 % F-12 (Gibco), 10 % fetal calf serum (Integro), 1 % L-glutamine, 100 U penicillin ml–1 and 0.1 mg streptomycin ml–1 at 37 °C in a 5 % CO2 atmosphere.
Invasion of T84 cells by C. jejuni strains of poultry and human origin. T84 cells were seeded in 96-well plates at 1x105 cells per well. After 78 h, the cells were inoculated with bacterial cells grown overnight in broth without horse blood at an m.o.i. of 200. The plates were centrifuged at 500 g for 10 min at 37 °C to enhance bacterial contact with the cell monolayer. The plates were incubated for 3 h at 37 °C in a 5 % CO2 atmosphere. Cells were washed with Hanks' Buffered Salt Solution (HBSS; Gibco) with Ca2+ and Mg2+ to remove non-invaded bacteria. To kill extracellular bacteria, 100 µg gentamicin ml–1 (Gibco) was added to the wells. This concentration proved to be lethal for all strains tested. Plates were further incubated for 2 h at 37 °C in a 5 % CO2 atmosphere. Gentamicin was washed away with HBSS without Ca2+ and Mg2+. Intracellular bacteria were released from T84 cells with 0.25 % sodium deoxycholate and titrated on mCCDA plates.
CDT production by poultry and human isolates. The CDT production of C. jejuni isolates was determined by the assessment of the number of HEp-2 cells blocked in the G2 phase after exposure to C. jejuni lysate (Young et al., 2000). The DNA content of HEp-2 cells incubated with the Campylobacter cell sonicates was determined quantitatively by flow cytometry. HEp-2 cells were seeded at a density of 1x105 cells ml–1 in 25 ml culture flasks. The cells were treated with 100 µl bacterial sonicate and incubated for 72 h at 37 °C with 5 % CO2. Sonicates from C. jejuni NCTC 11168 were used as a positive control (Purdy et al., 2000). Negative controls were treated likewise, but incubated with 100 µl PBS. After incubation, cells were washed once with medium without antibiotics and trypsinized. The cells were collected in a Falcon tube and pelleted by centrifugation at 1000 g for 5 min at 37 °C. DNA was stained according to Chien et al. (2000). Briefly, the pellet was resuspended in 0.5 ml staining solution (3 % PEG, 4 µg propidium iodide ml–1, 9 U RNase A ml–1, 0.1 % Triton X-100, 0.0001 % BSA, in 4 mM sodium citrate) for 20 min at 37 °C, followed by the addition of 0.5 ml salt solution (3 % PEG; 4 µg propidium iodide ml–1, 9 U RNase A ml–1, 0.1 % Triton X-100, 0.0001 % BSA, in 0.4 M NaCl) and the mixture was stored for 1.5 h at 4 °C in the dark. The DNA content was analysed using a FACSCalibur flow cytometer (Becton-Dickinson) and data acquisition was performed using CellQuest software. For each experiment, 104 cells were counted.
Presence of the virB11, cdtA, cdtB and cdtC genes and the CmeABC multidrug efflux pump in human and poultry isolates of C. jejuni. The presence of the virB11 gene as a marker for the plasmid pVir, the genes involved in CDT production cdtA, cdtB and cdtC, and the cmeABC genes for the multidrug efflux pump, was assessed using PCR. Bacterial chromosomal DNA was isolated from an overnight liquid culture without horse blood, using the DNeasy Tissue kit (Qiagen) according to the manufacturer's instructions.
Primers (Operon Biotechnologies) used in this study and their sources are listed in Table 1
. PCR was performed in a volume of 10 µl containing 0.03 U µl–1 Polymerase Taq Platinum, 1x PCR buffer, 3 mM MgCl2 (Invitrogen Life Technologies), 40 µM dNTP (Qiagen), 0.5 pmol µl–1 primers and 20 ng genomic DNA. Annealing temperature conditions varied according to the set of primers used. PCR products (3 µl) were separated by electrophoresis in an agarose gel containing 1.5 % agarose (Boehringer Mannheim) in 1x Tris-acetate/EDTA (TAE) buffer, pH 8, and stained with ethidium bromide. Gels were visualized using Image Master VDS (Pharmacia Biotech).
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Induction of IL-8 production by T84 cells exposed to C. jejuni strains of poultry and human origin.
T84 cells were seeded in 96-well plates at a concentration of 1x105 cells per well. After 78 h the cells were inoculated with a bacterial culture grown overnight in broth without horse blood at an m.o.i. of 200. Wells were incubated with PBS as the negative control and with tumour necrosis factor (TNF)-
(200 ng ml–1; Sigma) as the positive control. The plate was centrifuged at 500 g for 10 min to facilitate contact of bacteria with cells, and incubated for 24 h at 37 °C and 5 % CO2. Supernatant was collected and centrifuged (2300 g, 10 min, 4 °C). The concentration of IL-8 in cell culture supernatant was measured by an ELISA (Amersham Biosciences) according to the manufacturer's instructions.
Statistical analysis. Differences in occurrence of the virB11 gene in both populations were evaluated by means of Fisher's exact test. Differences in T84 invasion, HEp-2 cell cycle arrest and IL-8 production were tested by means of one-way ANOVA. For the T84 results, log10 transformation was performed to obtain normally distributed data. The capacity of the different strains to survive on different concentrations of sodium deoxycholate was analysed by means of Cox proportional hazard analyses. All statistical tests were performed in SPSS 14.0.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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To test the genetic stability of the isolates during passage, nine poultry isolates and nine clinical isolates recovered from cloacal swabs after passage were compared by PFGE to the original isolates that were used for oral inoculation of the chick. It was confirmed that six poultry isolates recovered after passage showed 100 % similarity to the original isolates by PFGE, while two isolates were slightly different (>90 % similarity) and one isolate was somewhat more different (80 % similarity). Four of the nine clinical isolates recovered after passage were identical to the original isolates by PFGE (>97 % similarity), while four isolates were slightly different (>92 % similarity) and one strain was more different (67 % similarity).
The 44 C. jejuni isolates tested in this study belonged to a large variety of genetic types, as none of the strains was identical as shown by PFGE. A certain degree of genetic instability was observed with PFGE after passage through day-old chicks for some of these strains, confirming earlier observations with passage through chick intestines (Hänninen et al., 1999).
Prevalence of the virB11 gene is equal among poultry and human isolates
Since the virB11 gene is associated with invasiveness (Bacon et al., 2000), we screened the C. jejuni collection for its presence using PCR. The virB11 gene was present equally often in the poultry strains as in the human strains (P=0.20), suggesting that some poultry isolates are capable of invading the human intestine. It should be noted, however, that the prevalence of the virB11 gene was low: the virB11 gene was present in only six of the 24 human isolates and two of the 20 poultry isolates.
C. jejuni strains from poultry and humans invade T84 cells equally
Invasion is generally considered to be important in pathogenesis, and a higher invasive capacity is generally reported for clinical isolates than non-clinical isolates (Fauchere et al., 1986; Konkel & Joens, 1989; Prasad et al., 1996; Tay et al., 1996). In our study, a total of 20 strains of poultry origin and 24 strains of human origin were compared with respect to their ability to invade T84 monolayers. Strains were considered invasive from log10(c.f.u. ml–1)=1.0 and above. Data from four independent experiments for the poultry isolates and three independent experiments for human clinical isolates are summarized in Table 2. All but one isolate from poultry and one isolate of human origin were invasive. There was no significant difference (P=0.48) in mean invasiveness between the poultry isolates [mean±SEM log10(c.f.u. ml–1)=2.5±0.1] and the human clinical isolates [mean±SEM log10(c.f.u. ml–1)=2.7±0.1], supporting the findings of Lindblom & Kaijser (1995). However, strong differences in invasiveness between individual strains were noticed, and a greater proportion of human isolates than poultry isolates was highly invasive with log10(c.f.u. ml–1) >3.5, but this tendency was not statistically significant.
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), confirming the findings on differences in toxigenicity between clinical and poultry isolates of Gilbert & Slavik (2004) and Prasad et al. (1996). For the extreme values, no differences in proportions between the poultry isolates and the human isolates could be found: one isolate from poultry (5.3 %) and two isolates from humans (8.7 %) were considered to be negative in their ability to produce functional CDT, while four poultry isolates (21.1 %) and seven human isolates (30.4 %) arrested more than 70 % of the HEp-2 cells in their cell cycle progression. The proportion of human isolates (34.8 %) which could induce between 50.0 and 70.0 % of the HEp-2 cells to remain locked in the cell cycle was greater than the equivalent proportion of poultry isolates (5.3 %).
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C. jejuni isolates from humans are more resistant to deoxycholate than poultry isolates
The ability of C. jejuni to reach the intestinal tract is associated with gastric-acid and bile-salt tolerance, and is a prerequisite for successful colonization and thus the induction of disease. Lin et al. (2003) showed a relationship between bile-salt resistance, colonization ability and the expression of the cmeABC operon. In our study, the prevalence of the cmeABC complex among the C. jejuni isolates was assessed using PCR. Although all 44 strains examined in this study possessed the three genes, strains were markedly different in their capacity to survive different concentrations of sodium deoxycholate. Human isolates appeared to be significantly more resistant to bile salts than the poultry isolates (P=0.006) (Fig. 1), with 85 % of the human strains surviving 16 000 µg sodium deoxycholate ml–1 compared to 54 % of the poultry isolates. Increased bile-salt tolerance might thus predispose to clinical campylobacteriosis in humans.
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. Strains inducing more than the mean of the negative control plus 2x SEM (346.0 pg ml–1) were considered to be positive in their ability to induce IL-8 production. The positive TNF- (200 ng ml–1) control induced a mean of 899.3±57.0 pg IL-8 ml–1. All strains mediated IL-8 production from the T84 monolayers. Although isolates of poultry origin (n=18; mean±SEM 643.9±31.3 pg ml–1) were not statistically different (P=0.29) in their ability to induce IL-8 production from the human clinical isolates (n=24; mean±SEM 672.6±23.5 pg ml–1), it was notable that more human isolates (66.7 %) induced an IL-8 response between 600 and 700 pg ml–1 than did poultry isolates (33.3 %). In both C. jejuni populations, highly specialized strains exist: the lysate of the poultry strain KC 44 arrests only a small percentage of HEp-2 cells and is a poor invader, but causes the highest IL-8 response. Likewise, the human strain R-27478 induces an IL-8 response, but is a low CDT producer and invades T84 cells poorly. In contrast, two highly invasive and CDT-producing strains elicit the IL-8 response only moderately.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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