1 National Veterinary Institute, Technical University of Denmark2 Division of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark3 Section of Poultry Diseases, Division of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark4 Division of Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark5 Department of Micro- and Nanotechnology, Technical University of Denmark6 BioLabChip, Department of Micro- and Nanotechnology, Technical University of Denmark7 National Food Institute, Technical University of Denmark
Campylobacter is the most common cause of food-borne illness in Europe, and this important zoonotic pathogen has been the focus of many research projects and scientific publications in recent years. However, we know less about the biology and pathogenicity of this pathogen than we know about many less prevalent pathogens. In this PhD project, I have investigated the survival and virulence of Campylobacter spp. in various matrices such as chicken faeces, swine manure and in co-culture with protozoa. In the first study, using bacterial culture and RT-qPCR methods, I found that viable C. jejuni cells could be detected for up to 5 days in both spiked and the naturally Campylobacter contaminated chicken faecal samples. Negative RT-qPCR was obtained when viable C. jejuni cells could not be counted by culture. In contrast, using a DNA-based qPCR method, dead or non-viable Campylobacter cells were detected, since all tested samples were positive, even after 20 days of storage. In the second study, the survival of C. coli in swine manure during storage for 30 days was studied by three different methods: bacterial culture (plate counting), DNA qPCR, and RT-qPCR. I found that C. coli could survive in swine manure up to 24 days at 4°C. At higher temperatures, this bacterium survived only 7 days (15°C) or 6 days (22°C) of storage. The survival of C. coli was extremely short (few hours) in samples incubated at 42 and 52°C. I also found that the RT-qPCR method not only can detect and differentiate living bacteria from dead cells, but also can be used to study the survival and potential pathogenicity of bacteria based on expression of different virulence genes. In a collaborated study, I have investigated the leaching potentials of a Salmonella Typhimurium phage type 28B and two bacteria: Escherichia coli and Enterococcus spp., in raw slurry, in the liquid fraction of separated slurry, and in the liquid fraction after ozonation to ground water using intact soil columns models. I observed that solid-liquid separation of slurry increased the redistribution of contaminants in liquid fraction in the soil compared to raw slurry, and the recovery of E. coli and Enterococcus spp. was higher for liquid fraction after the four leaching events. The liquid fraction also resulted in a higher leaching of all contaminants except Enterococcus spp. than raw slurry while the Ozonation reduced E. coli leaching only. Protozoa including amoebae have been found widely in broiler houses. It has been shown that freeliving protozoa may harbor, protect, and disperse bacteria, including those ingested and passed in viable form in feaces. Therefore it is very interesting to study their role in the survival of Campylobacter. In the second part of my PhD project, I have investigated the mechanisms involved in the interactions of Campylobacter and two protozoa: Acanthamoeba castellanii and Cercomonas sp. which are commonly found in soil and water. I have found that C. jejuni can survive intracellularly within A. castellanii for a short time (5 h after gentamicin treatment) at 25ºC in aerobic conditions. Conversely, I found that A. castellanii promoted the extracellular growth of C. jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae – bacteria contact. Interestingly, I identified the depletion of dissolved oxygen by A. castellanii as the major contributor for the observed amoeba-mediated growth enhancement. To test whether another protozoan rather than Acanthamoeba has similar impacts on survival of C. jejuni as well as other food-borne pathogens S. Typhimurium and Listeria monocytogenesis, I have investigated the interactions between a common soil flagellate, Cercomonas sp., and these three bacterial pathogens. I observed a rapid growth of flagellate in co-culture with C. jejuni and S. Typhimurium over the time course of 15 days. In contrast, the number of Cercomonas sp. cells decreased when grown with or without L. monocytogenes for 9 days of co-culture. Interestingly, I observed that C. jejuni and S. Typhimurium survived better when co-cultured with flagellates than when cultured alone. The results of this study suggest that Cercomonas sp. and perhaps other soil flagellates may play a role for the survival of these food-borne pathogens on plant surfaces and in soil. It would be very interesting to further investigate the impacts of this soil flagellate on the survival of different food-borne pathogens in soil and in plant surface that may explain the epidemiology of recent outbreaks of food-borne diseases from vegetables. During transmission and infection, C. jejuni may encounter many different stresses but little is known about how this bacterium survives and interacts with the protozoa under these conditions. I have investigated the impacts of environmental stress factors, namely heat shock, starvation, osmosis, and oxidation, on the expression of three virulence genes (ciaB, dnaJ, and htrA) of C. jejuni and its uptake by and intracellular survival within A. castellanii. I also investigated the mechanism(s) involved in phagocytosis and killing of C. jejuni by A. castellanii. I observed that heat and osmotic stresses reduced the survival of C. jejuni significantly, whereas oxidative stress had no effect. The results of qRT-PCR experiments showed that the transcription of virulence genes of C. jejuni was slightly up-regulated under heat and oxidative stresses but down-regulated under low nutrient and osmotic stresses, the htrA gene showing the largest down-regulation in response to osmotic stress. The results also showed that C. jejuni rapidly loses viability during its intra-amoeba stage and that exposure of C. jejuni to environmental stresses did not promote its intracellular survival in A. castellanii. In addition, the results indicated that this bacterium uses a distinct strategy for phagocytosis which involves recruiting actin for internalization in the absence of PI 3-kinasemediated signal. The studies also identified that phago-lysosome maturation may not be the primary factor for intra-amoeba killing of C. jejuni. Together these findings suggest that the stress response in C. jejuni and its interaction with A. castellanii are complex and appear multifactorial.
C. jejuni; C. coli; L. monocytogenes; S. Typhimurium; Flagellate; Cercomonas sp.; Acanthamoeba castellanii; Manure separation; Groundwater contamination; RT-qPCR; Environmental stresses; Virulence; Chicken faeces