Increase in Acid Tolerance of Campylobacter jejuni through Coincubation with Amoebae

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga. Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.Campylobacter jejuni is a major cause of human bacterial enteritis, with an incidence exceeding that of Salmonella spp. or Escherichia coli O157 (6, 28). Most infections are associated with consumption of contaminated food, primarily undercooked chicken meat, but unchlorinated water and unpasteurized milk can also be sources of Campylobacter infection (reviewed in reference 13). Apart from food-borne sources, additional risk factors include close contact with pets or farm animals and activities in recreational waters (reviewed in reference 13). C. jejuni is widely distributed in many animals and has also been reported to be isolated from surface waters (15) and, occasionally, even from groundwater (31). However, the bacterium has been shown to be relatively sensitive to environmental stress outside its hosts, including heating, disinfectants, oxygen exposure, osmotic stress, desiccation, and acidity (5, 9, 19, 35).Several hygiene practices have been implemented in broiler production facilities to reduce C. jejuni carriage in live birds. Such measures include hygiene barriers such as changing clothes before entering the broiler houses and disinfection of the interior of the building with acid between flock rotations (20). Such efforts may reduce the number of C. jejuni organisms, but the bacterium is still difficult to eradicate from contaminated farms, and subsequent outbreaks at the same farm are not rare (11). Contradictory to its fragility in different in vitro settings, C. jejuni seems to be well adapted to survive the acidic milieu of the human stomach during the passage to the lower intestinal tract, where infection is established. This is illustrated by the very low infectious dose for both broiler chickens (7) and humans (4) and indicates that the bacterium has developed strategies to avoid or withstand low pH in order to survive the transit. The gastric acid is the first line of defense against ingested pathogens. During fasting conditions in healthy humans, the luminal pH in the stomach is usually around 2.0, but it may range from 1.5 to 5.5 depending on food intake, such as a diet with a high pH, or the use of proton pump inhibitors (36). Laboratory studies have demonstrated that C. jejuni in solution survives a maximum of 30 min at pH levels below pH 2.5 and for up to 60 min at pH 3 (5, 23). When the bacterium is mixed with food, it seems to be protected, and it has been shown that C. jejuni inoculated onto ground beef survived at pH 2.5 for 2 h at 37°C (37).In the last few years, laboratory studies have identified a new potential epidemiological pathway for C. jejuni in which the bacterium colonizes unicellular eukaryotic organisms (protozoa) and thereby acquires protection from adverse environmental conditions (2, 17, 29). C. jejuni can colonize protozoa and survive longer in its protozoan host than as a free-living bacterium, and given the right temperature, the bacterium can also replicate intracellularly (1, 2). Protozoa, especially amoebae, serve as natural reservoirs or vehicles for the dissemination of several other pathogenic bacteria, including Legionella pneumophila (25), Vibrio cholerae (34), and Helicobacter pylori (38). Amoebae are abundant in virtually all natural water systems and can be found grazing on biofilms in water supply systems (14). In their trophozoite form, amoebae are naturally resistant to many environmental factors that are lethal to Campylobacter, and they can multiply at pHs ranging from 4 to 12 (16). Moreover, amoebae can enter a cyst form when challenged with unfavorable conditions. These cysts generally have a double cell wall that might explain their capability to survive chlorination, antimicrobials, and changes in pH and osmotic pressure. This resistance feature of amoebae makes them suitable hosts for other, less-resistant microorganisms (16, 32).In this study, we built on the advances gained in protozoa-Campylobacter research and investigated whether internalization of C. jejuni into Acanthamoeba affects bacterial tolerance to hydrochloric acid. Using an in vitro setup, we found that C. jejuni survived better in an acidic environment when it was coincubated with amoebae than when it was incubated as bacteria in solution. Furthermore, we show that bacterial motility and adhesion to and internalization into amoeba are trigged by moderately acidic conditions. The implications of these findings for the survival of C. jejuni in food production as well as in transit through the human stomach are discussed.