Influence of Adaptive Tolerance Response and Quorum Sensing on Virulence and Pathogenicity of Human and Poultry Isolates of Campylobacter Jejuni

<p>NON-TECHNICAL SUMMARY:<br/> Bacterial pathogens have been found to adapt to stresses used in processing and survive by acquiring an adaptive tolerance response. Recent findings indicate that foodborne pathogens such as Salmonella, Listeria and E. coli are capable of surviving stressful conditions by the mechanism of adaptive tolerance response (ATR). Stress adaptation can cause potential health hazard to humans as the stress-adapted bacterial cells can become more resistant to cooking and other methods commonly used to achieve pathogen reduction. Even though the fragile C. jejuni requires fastidious growth conditions in the laboratory, it is able to persist and survive in the environment and continue to be a serious public threat. Compared to other foodborne bacterial pathogens not much is known about the mechanisms by which C. jejuni survive stressful conditions. Recent
studies show that C. jejuni also possesses an adaptive tolerance response which helps its survival in adverse environments. Knowledge about the mechanisms helping this organism to produce an adaptive response mechanism can help to assess and strengthen intervention methods in poultry processing plants and, thus, reduce/ eliminate the organism in poultry, which ultimately leads to reduced numbers of human foodborne illnesses.<p>
APPROACH: <br/>Objective 1. Natural plant extracts such as thymol and carvacrol were found to reduce C. jejuni colonization in birds (Arsi et al., 2010). To determine the effects of plant extracts on C. jejuni, various strains of C. jejuni will be treated with plant extracts such as thymol, carvacrol, eugenol, trans-cinnamaldehyde and beta-resorcylic acid in vitro and in vivo on poultry meat and survivability of C. jejuni will be determined. Similarly the effects of stresses such as acid, salt, high and low temperatures, starvation and oxidative stress will be evaluated using different strains of C. jejuni. Exposure of bacteria to sub-lethal stresses induces an adaptive tolerance response (ATR) which provides protection towards subsequent exposure to a lethal stress. To determine the ATR of the C. jejuni isolates, they will be subjected to different mild stresses and
subsequently exposed to severe secondary stresses. Isolates from working cultures will be grown in Campylobacter enrichment broth for 18 h, centrifuged at 3000 x g for 5 minutes, supernatant discarded and the pellet re-suspended in acid broth pH 5.5 (mild acid), phosphate buffered saline pH 7.2 (starvation) or salt (1%) to obtain the respective stress-adapted cells. The stress-adapted cells will further be subjected to the same stress or different stresses such as acid broth (pH4.5), starvation, salt (3%) or high temperature (60oC)and the adaptive ability of the C. jejuni will be measured by taking samples at different times during the experiment to construct a survival plot. Viable cell counts will be estimated before and after stress by plating serial dilutions of each culture on Campylobacter blood agar plates. By comparing the survival plot of stressed and non-stressed cells, the
adaptation of C. jejuni to different stresses will be determined. Objective 2. The pathogenesis of Campylobacter infection is still not very clear. In humans, once C. jejuni cells are ingested via contaminated food or water and reach the intestine, they attack the mucosal layer of the intestines. Adhesion and invasion of the intestinal epithelial cells by C. jejuni are two important steps in producing the disease. Experiments in our laboratory have proven that adhesion and invasion of the tissue culture cells (INT 407 cells) by C. jejuni in vitro is similar to that happening in vivo in humans. INT 407 cells are derived from human embryonic intestinal cells and will be used as an in vitro model to determine attachment and penetration of stress-adapted C. jejuni versus non-stress-adapted cells. The tissue culture cells will be grown in Eagle's Basal Medium with 10% fetal bovine serum
and 2mM L-glutamine in 75 cm2 tissue culture flasks at 37?C in a 5% CO2 atmosphere. Intestinal cells will be seeded onto 24-well tissue culture plates at a concentration of 105 cells per well and plates incubated for 24 h to allow cell attachment. After 24 h, wells will be washed 3 times with sterile PBS. Stress-adapted and non-stress adapted bacterial cells exposed to further stress will be used to infect the wells at 1 mL per well. Plates will be incubated for 2 h to allow adherence and invasion of tissue culture cells. After 2 h, all plates will be washed three times with PBS to remove any non-adhering bacteria before the addition of fresh medium containing 100?g ml-1 gentamicin (to measure invasion alone) or, in the duplicate plates, PBS with 0.1% Triton X-100 (to measure adherence and invasion). Plates with media plus gentamicin will be incubated for an additional 2 h, while
plates with PBS plus 0.1% Triton will be gently swirled for 30 minutes at room temperature to lyse the cells and release internalized bacteria. After the 2 h incubation of plates with media plus gentamicin, plates will be washed two times with PBS prior to the addition of PBS with 0.1% Triton X-100. Cells will be lysed using the same method as described above. To determine the colony forming units (cfu) of bacteria adhering and invading the tissue culture models, ten-fold dilutions will be made of the lysed cells in sterile PBS and plated onto CE agar. Objective 3. It is assumed that all pathogenic bacteria will exhibit an increased expression of their virulence genes when they are subjected to stress conditions, which helps them in their survival. To measure any changes in virulence gene expression in stress-adapted C. jejuni, RNA will be collected from stress-adapted and non-stress
adapted cells after their exposure to a subsequent stress as we have previously described (Ma et al., 2009). Primers specific for virulence genes of C .jejuni including ciaB, cdtB, flaA, cadF, and sodB will be used as described in our previous studies (Ma et al., 2009 and Hanning et al., 2009). RT-PCR will be conducted with the one-step quantitative real-time RT-PCR kit with SYBR green. After each amplification, melting curve analysis will be performed and the relative changes (n-fold) in virulence gene transcription will be calculated using the 2-CT method as described by Livak and Schmittgen (2001). In order to identify the stress-induced proteins, we will combine two-dimensional gel electrophoresis and mass spectrometry to perform proteomic analysis. Late exponential cells of C.jejuni will be harvested and the protein extracted and subjected to the two-dimensional gel electrophoresis.
The two-dimensional gel electrophoresis will be performed as described in a previous study (Ma et al., 2009). Protein spots with altered levels of expression under different stress conditions will be excised from the gel, using the ProPick excision robot (Genomic Solutions), and in-gel trypsin digested using a ProGest Protein Digester (Genomic Solutions). The tryptic digests will be analysed using a Reflex III MALDI-TOF instrument (Bruker) with a Scout 384 ion source using a nitrogen laser (wavelength 337nm) to desorb/ionize the matrix/analyte material from the sample substrate. Mascot search tool (Matrix Science) will be used to identify proteins from the peptide mass peak list by the Protein Mass Fingerprint technique. Then, the C.jejuni protein sequences in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/) will be searched to identify the
stress-induced proteins. Objective 4. Communication between bacteria, generally referred to as quorum sensing allows bacteria to regulate their genes and co-ordinate their behavior as a group in response to any changes in the environment. To study the effects of quorum sensing on adaptive tolerance response of C. jejuni, 2 isolates of C. jejuni will be used: (1) the human isolate 81176 and (2) a luxS mutant of the human isolate 81176, which will be used as the control. AI-2 production in C. jejuni is dependent on the product of luxS gene (Elvers and Park, 2002). Viable cell counts of both the mutant strain and the 81176 isolate will be determined after exposure to different stresses using the same protocol as described in objective a. Adhesion and invasion assays will be performed as described in objective c to determine the effects of AI-2 on adhesion and invasion of intestinal cells by
C. jejuni.</p>