Increase in Acid Tolerance of Campylobacter jejuni through ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2010, p. 4194–4200 0099-2240/10/$12.00 doi:10.1128/AEM.01219-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 13

Increase in Acid Tolerance of Campylobacter jejuni through Coincubation with Amoebae䌤 Diana Axelsson-Olsson,1†* Lovisa Svensson,1† Jenny Olofsson,1,2 Paulo Salomon,4 Jonas Waldenstro ¨m,1,2 Patrik Ellstro ¨m,3 and Bjo ¨rn Olsen1,2 Section for Zoonotic Ecology and Epidemiology, Linnaeus University, SE-391 82 Kalmar, Sweden1; Section of Infectious Diseases, Department of Medical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden2; Clinical Bacteriology, Department of Medical Sciences, University of Uppsala, SE-751 85 Uppsala, Sweden3; and Marine Sciences Division, Linnaeus University, SE-391 82 Kalmar, Sweden4 Received 28 May 2009/Accepted 25 April 2010

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. taminated 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

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 con-

* Corresponding author. Mailing address: Section for Zoonotic Ecology and Epidemiology, Linnaeus University, SE-391 82 Kalmar, Sweden. Phone: 46-480-446229. Fax: 46-480-447305. E-mail: diana [email protected]. † Diana Axelsson-Olsson and Lovisa Svensson contributed equally to this work. 䌤 Published ahead of print on 7 May 2010. 4194

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ACID TOLERANCE OF C. JEJUNI ASSOCIATED WITH AMOEBAE

(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 protozoaCampylobacter 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. MATERIALS AND METHODS Bacterial and amoebic cultures. Campylobacter jejuni CCUG 11284 was used in all experiments. This reference strain was originally isolated from bovine feces and has previously been used to study Acanthamoeba-Campylobacter interactions (1, 2). Before each experiment, bacteria were grown on conventional blood agar plates (Columbia agar II containing 8% [vol/vol] whole horse blood) at 42°C for 24 h in a microaerobic environment, using a CampyGen gas-generating system (CN0025A; Oxoid, Ltd., Basingstoke, United Kingdom) and a BBL GasPak system (BD, Franklin Lakes, NJ). Bacterial cells were harvested and diluted in a peptone-yeast extract-glucose (PYG) medium (24), with later modifications of Greub and Raoult (10), to a final concentration of 105 to 107 CFU/ml (estimated using plate counts) and were used as a stock solution. Three species of amoebae were used in the challenge experiments: Acanthamoeba polyphaga (strain Linc Ap-1), Acanthamoeba castellanii, and Acanthamoeba rhysodes. The last two were isolated from Swedish patients with keratitis and were provided by J. Winiecka-Krusnell (Karolinska Institute, Sweden). Stock cultures of each Acanthamoeba species were maintained in a PYG medium at 27°C in 25-cm2 culture flasks (Sarstedt, Nu ¨rnbrecht, Germany) (2). For the experiments, Acanthamoeba suspensions in PYG were transferred to 12-well culture plates (1 ml/well) and incubated at 27°C for 3 days, until the cells formed confluent cell layers in the bottom of the wells. Acidified test medium. Phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO) and tap water from a private well (from the village of Ramsås, Sweden), supplemented with modified Bolton broth selective supplement (SR0208; Oxoid), were used as test media. The water was analyzed for ion content (2.9° dH), conductivity (14 mS/m), and pH (7.0) (Eurofins, Kalmar, Sweden). For each of the experiments below, PBS and water were autoclaved and aliquoted in four 50-ml tubes each. Four different pH levels per medium type and experiment were prepared by using hydrochloric acid (HCl) as an acidifier to pH 1, 2, 3, 4, and 5, with an acceptance level of ⫾0.05 pH unit. For the experiment with different Acanthamoeba species, pH 1 was adjusted to pH 1.5. The pH level of the fluid in each well was continuously measured during the survival experiments, and an increase in initial pH 3 and 4 to pH 4 and 5 was observed. Therefore, pH 3 and pH 4 are referred to as pH 4 and pH 5, respectively. Survival of C. jejuni cells coincubated with A. polyphaga amoebae in acidified medium. To test whether the presence of amoebae in the medium influenced the survival of C. jejuni at different levels of acidity (pH 1, 2, 4, and 5), we used two acidified test media (water and PBS) and the following three treatments: C. jejuni preincubated with A. polyphaga before acid treatment (treatment A), C. jejuni mixed with A. polyphaga after the onset of acid treatment (treatment B), and C. jejuni in acidified medium without A. polyphaga (treatment C). For treatment A, 12-well plates with confluent A. polyphaga layers in PYG medium were seeded with C. jejuni in each well. These plates were incubated for

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2 h at 32°C to allow the bacterial cells time for colonization of the A. polyphaga amoebae. The PYG medium was gently removed and replaced by 3 ml of acidified water or acidified PBS at the different pH levels. For treatment B, two additional plates with confluent A. polyphaga amoebae were prepared by gently removing the PYG medium and replacing it with 3 ml acidified water or acidified PBS. For the control treatment (treatment C), plates without amoebae were prepared with identical pH and medium combinations. For treatments B and C, plates were seeded with bacteria following the pH change. All experiments were done in triplicate in 12-well culture plates, resulting in three similar wells for each combination of pH and medium. All plates, regardless of treatment, were then incubated at 32°C in an aerobic environment. From each of the 72 treated wells, a 100-␮l sample was taken at time zero and at 5 and 20 h. The samples were 10-fold serially diluted in PYG medium and spread on blood agar for colony counting. For each well and each time point, a measure of C. jejuni cell survival was calculated by dividing the bacterial concentration of the sample (estimated from colony counts) by the bacterial concentration of that well at time zero. Differences in bacterial survival among treatments were calculated separately for each combination of pH and medium by comparing the distributions of survival estimates (three replicates per experiment and three independent experiments) by the Kruskal-Wallis nonparametric test, considering P values of ⬍0.05 to have statistical significance. Importance of Acanthamoeba species for C. jejuni survival at low pH. An additional experiment was set up to investigate whether promotion of C. jejuni survival was independent of which Acanthamoeba species was used. Therefore, C. jejuni survival was compared between coincubations with A. polyphaga, A. castellanii, and A. rhysodes. The setup was the same as in the previous experiment, regarding treatments, media, and pH levels, except that pH 1 was adjusted to pH 1.5. Instead of a quantitative estimation, the survival was determined by growth versus nongrowth of C. jejuni on blood agar plates. The samples were scored at 0, 1, 2, 4, 5, 20, and 40 h of incubation. Viability and morphology of A. polyphaga in response to acidic conditions. The viability of A. polyphaga was examined using the trypan blue exclusion test. A. polyphaga cultures were incubated for 24 h in PBS at pH 2, 4, or 7. The cells were then gently detached by repeated pipetting and were mixed with 0.04% trypan blue (Sigma-Aldrich, St. Louis, MO). The viability was determined by calculating the ratio of stained versus unstained cells in a Bu ¨rcher chamber. Effect of pH on adhesion/internalization of C. jejuni into A. polyphaga. The adhesion/internalization of C. jejuni into A. polyphaga at pH 2, 3, 4, 5, and 7 was quantified by flow cytometry and determined manually by assessing the amoebaassociated bacteria by fluorescence microscopy (Axioskop microscope; Zeiss, Go ¨ttingen, Germany). Staining of the bacteria was performed using a 50 mM cyanoditolyl tetrazolium chloride (CTC; Polyscience, Eppelheim, Germany) solution according to the manufacturer’s instructions, with incubation for 1 h at room temperature in the dark. Bacterial cells were washed once in PBS and centrifuged at 3,000 ⫻ g for 10 min. The pellet was resuspended in either PBS with pH 7 or acidified PBS at pH 2, 3, 4, or 5. A. polyphaga amoebae were cultured in 75-cm2 culture flasks (Sarstedt) until confluent, washed once with PBS, and detached by incubation at ⫺20°C for 10 min. The cells were transferred to 15-ml Falcon tubes and washed twice in PBS by centrifugation at 400 ⫻ g for 8 min. Prior to viable counts as previously described, the cell suspension was filtered through a 50-␮m-mesh nylon net to remove large particles that could clog the sample line in the flow cytometer. The pH was changed by washing the trophozoites twice in acidified PBS. A. polyphaga amoebae, with or without CTC-stained C. jejuni (infection ratio, 1:100), were incubated in 15-ml tubes with end-over-end rotation for 1 h at room temperature in the dark. Unbound extracellular bacteria were removed, and the pH was restored to neutral by two washes in PBS (pH 7). Cell suspensions (1 ml) were transferred to 5-ml PE Falcon tubes and were analyzed by flow cytometry (FACSCalibur, Becton Dickinson, CA), using the blue laser line (488 nm) for excitation. Data acquisition and analysis were done with CellQuest v. 3.3 software. The flow cytometer was set up to detect A. polyphaga cells in a forward (FSC) versus side scatter (SSC) plot, with both axes on a logarithmic scale. FSC was used as the threshold triggering parameter. The A. polyphaga population was then marked in the FSC versus SSC plot and gated into a histogram of the red fluorescence signal (FL3) collected through a 650-nm-long band-pass filter, on a logarithmic scale. A minimum of 10,000 A. polyphaga cells were analyzed in each sample. The geometric mean fluorescence intensity (MFI) of the amoeba cell population was determined from the histograms. Incubation of A. polyphaga amoebae at low pH gave rise to red autofluorescence. Therefore, the MFI ratio of each sample was calculated by dividing the MFI of amoebae coincubated with bacteria by the MFI for the corresponding sample of amoebae without bacteria

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FIG. 1. Survival of C. jejuni in water at pH 5 (a) and pH 4 (b) and in PBS at pH 5 (c) and pH 4 (d). The box plots show survival as the % of initial CFU after 5 h and 20 h. Data are based on three independent experiments with C. jejuni treated in three different ways: treatment A (white boxes), C. jejuni added prior to acidification; treatment B (striped boxes), C. jejuni added after acidification; and treatment C (dotted boxes), C. jejuni without A. polyphaga, as a control.

and treated at the same pH (coincubated/control). The data presented are from two independent observations. The same samples were also analyzed by fluorescence microscopy. For each sample, 100 amoebae were analyzed, and the number of C. jejuni cells adhered to or internalized in each amoeba was counted. With the setup used in this study, neither of the methods could conclusively discriminate between adhered and internalized bacteria, and therefore we use the term “adhered/internalized” to describe the interactions between C. jejuni and A. polyphaga. However, a substantial number of the CTC-stained C. jejuni cells could be found intracellularly, as determined by the fact that they were clearly seen inside vacuoles. Data were collected from three independent observations and are shown as means ⫾ standard deviations (SD). For statistical analysis, one-way analysis of variance (ANOVA) was used, followed by Tukey’s test, to compare samples in acidified PBS to samples in PBS, pH 7. Effect of pH on C. jejuni motility. A swarming assay was performed on thioglycolate soft agar (0.4% [wt/vol]) plates adjusted to pH 3, 4, 5, and 7. The assay was slightly modified from the method of Szymanski and colleagues (33). Five microliters of C. jejuni stock solution or C. jejuni preexposed to pH 1.5, 2, 3, 4, or 5 for 1 h at room temperature was inoculated onto an agar plate and incubated at 37°C for 48 h. The diameters of swarms were measured, and the mean of six

replicates was calculated for each pH level. The experiment was repeated three times. Statistical analysis was performed using Student’s paired t test to compare the values to swarming at pH 7. Data are shown as means ⫾ standard errors of the means (SEM).

RESULTS Survival of C. jejuni coincubated with A. polyphaga in acidified medium. Under moderately acidic conditions, the viability of C. jejuni was independent of treatment (treatments A, B, and C). At pH 5, ⬎60% of the bacteria survived after 5 h in water for treatments A, B, and C, with no significant differences between treatments (␹ ⫽ 3.65; df ⫽ 2; P ⫽ 0.16 [KruskalWallis test]) (Fig. 1a). However, after 20 h, there were pronounced differences in survival between bacteria coincubated with amoebae (treatments A and B) and the control bacteria (treatment C), particularly when the bacteria were added to

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amoebae after the pH change (treatment B). For this treatment (treatment B), 27.0% of the bacteria survived after 20 h in water, compared to 7.2% and 3.9% for treatments A and C, respectively (␹ ⫽ 9.87; df ⫽ 2; P ⫽ 0.007 [Kruskal-Wallis test]) (Fig. 1a). At pH 4, the differences between treatments were even more pronounced. With treatment B, 46.8% of the bacteria survived after 5 h in water, whereas 1.9% survived with treatment A and 6.3% survived with treatment C (␹ ⫽ 15.7; df ⫽ 2; P ⫽ ⬍ 0.001 [Kruskal-Wallis test]) (Fig. 1b). After 20 h at pH 4, only a few bacteria were viable. Still, there were significant differences between treatments (␹ ⫽ 13.3; df ⫽ 2; P ⫽ 0.001 [KruskalWallis test]), as a larger proportion of the bacteria had survived in water with treatment B (0.67%) than with treatment A (0.01%). In the control without amoebae, there were no viable bacteria after 20 h. Parallel experiments with identical setups were also performed with PBS as an acidified medium. Results at pH 4 and 5 were similar to the results obtained with water, and there were significant differences among treatments at both pH levels at 5 h (␹ ⬎ 10.7; df ⫽ 2; P ⬍ 0.005 [KruskalWallis test]) and 20 h (␹ ⬎ 14.1; df ⫽ 2; P ⬍ 0.001 [KruskalWallis test]) (Fig. 1c and d). At the lower pH levels, C. jejuni was detectable in water after 5 h with treatment A. The survival rates after 5 h were, on average, 5.0% at pH 1 and 10.5% at pH 2 (data not shown). No viable bacteria were detected after 20 h at pH 1 or 2, regardless of treatment. In summary, the results show that C. jejuni cells incubated in the presence of A. polyphaga survived longer at all pHs, but the differences were most pronounced at pH 4 and pH 5 compared to the control cells without amoebae. This effect was strongest when the bacteria were added to the protozoa after the pH change. Importance of Acanthamoeba species for C. jejuni survival at low pH. C. jejuni cells were coincubated with A. polyphaga, A. castellanii, and A. rhysodes amoebae at four different pH levels, pH 1.5, 2, 4, and 5. The survival of bacterial cells was compared after different time intervals. There were no differences in survival times of C. jejuni for cocultivation with A. castellanii, A. rhysodes, or A. polyphaga. The general trend was that C. jejuni cells coincubated with Acanthamoeba spp. survived longer than the control cells without amoebae at all pH levels tested (data not shown). Effect of pH on adhesion/internalization of C. jejuni into A. polyphaga. Since C. jejuni cells were better protected by A. polyphaga when the bacteria were added after acidification of the medium (treatment B) at pH 4 and pH 5, the effect of acidic conditions on bacterial association with amoebae was assessed. The results from the flow cytometric analysis showed an increase in the MFI of A. polyphaga cells infected with CTC-stained bacteria compared to that of control amoebae, suggesting bacterial adhesion/internalization into A. polyphaga (Fig. 2a). The MFI was augmented in a pH-dependent manner in amoebae coincubated with bacteria, and pH 4 and 5 gave the highest MFI (Fig. 2a). However, the MFI also increased with a lowered pH in controls (data not shown). This increase was due to red autofluorescence, and therefore we used the relative MFI, defined as the ratio of MFI (coincubated cells/control cells) (Fig. 2b). We found that at pH 4 and 5, the bacterial adhesion/internalization into A. polyphaga was increased compared to that at pH 7. However, at pH 2 and 3, there was no

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increase in bacterial adhesion/internalization, and instead, the MFI ratios were similar to those at pH 7 (Fig. 2b). To verify the flow cytometry data, the association of C. jejuni with A. polyphaga was manually quantified in a fluorescence microscope, which allowed both quantification of the number of bacteria associated with each amoeba and the percentage of amoeba cells with associated bacteria. The number of adherent/internalized bacteria per amoeba was shown to be dependent on the surrounding pH (F ⫽ 39.49; df ⫽ 4; P ⬍ 0.0001 [one-way ANOVA]) (Fig. 2c). For cells interacting under moderately acidic conditions (pH 4 to 5), adhesion/internalization increased significantly compared to that at pH 7. At pH 5, 6.65 ⫾ 0.94 bacteria were found inside or on the surface of each A. polyphaga trophozoite (P ⬍ 0.0001 [Tukey’s test]), and at pH 4, this number was 5.87 ⫾ 0.96 bacteria/amoeba (P ⬍ 0.0001 [Tukey’s test]) (Fig. 2c). Under more acidic conditions, the association of C. jejuni with A. polyphaga amounted to 2.23 ⫾ 0.54 bacteria/amoeba at pH 3 (P ⫽ 0.949 [Tukey’s test]) and 1.38 ⫾ 0.46 bacteria/amoeba at pH 2 (P ⫽ 0.949 [Tukey’s test]) (Fig. 2c). Under all of the different conditions, internalized and motile bacteria were observed in the vacuoles of A. polyphaga trophozoites. The percentage of amoebae associated with bacteria was also dependent on the surrounding pH (F ⫽ 13.78; df ⫽ 4; P ⬍ 0.0004 [one-way ANOVA]). At pH 7, 37.0% ⫾ 18% of A. polyphaga trophozoites had C. jejuni cells internalized or on the surface, compared to 78.0% ⫾ 12% at pH 5 (P ⫽ 0.016 [Tukey’s test]) and 80.0% ⫾ 8.7% at pH 4 (P ⫽ 0.012 [Tukey’s test]) (Fig. 2d). At pH 3 (36.7% ⫾ 5.5%) and 2 (21.0% ⫾ 15%), there was no significant difference in association compared to that at pH 7 (P ⫽ 1.000 and P ⫽ 0.547, respectively [Tukey’s test]) (Fig. 2d). In summary, both flow cytometry and microscopy data showed that the association of C. jejuni with A. polyphaga was increased at moderately low pH, suggesting that acidic conditions trigger such interactions. Effect of pH on C. jejuni motility. The effect of pH on the motility of C. jejuni was clearly pH dependent (Table 1). Swarms (1.5 ⫾ 0.03 cm) were detected on soft agar plates at pH 7, but C. jejuni cells were nonmotile on plates with a pH below 7 or when they were preexposed to extremely low pH levels (1.5 and 2). However, after pretreatment of C. jejuni cells with acidified PBS at pH 5 for 1 h, swarming was significantly increased compared to that of the control (1.9 ⫾ 0.03 cm; P ⬍ 0.001). At pH 4 and 3, motilities were similar to or somewhat attenuated compared to that at pH 7 (Table 1). Viability and morphology of Acanthamoeba in response to acidic conditions. After 24 h of exposure to pH 7, 84% of the trophozoites remained viable. Exposure to pH 4 did not result in decreased viability (86% after 24 h). However, after 24 h of incubation at pH 2, the viability had dropped to 13%. Observations in the microscope revealed that at pH 4 and 5, amoebae remained in the trophozoite form throughout the experiment, while at pH 1 to 1.5, they instantly changed morphology to a rounder shape and detached from the bottom of the wells, seemingly intact. Similar observations were made at pH 2, but with a delay of about 5 h. DISCUSSION Protozoa of the Acanthamoeba genus have been shown to facilitate intracellular survival and replication of C. jejuni (1, 2,

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FIG. 2. The effect of pH on adherence/internalization of C. jejuni into A. polyphaga was quantified using flow cytometry (a and b) and fluorescence microscopy (c and d). (a) Representative histograms of A. polyphaga alone at pH 7 (control) and A. polyphaga coincubated with CTC-stained C. jejuni at pH 2, 3, 4, 5, and 7. (b) The MFI ratio is given for coincubated/control amoebae at different pHs. (c) Number of adhered/internalized C. jejuni cells per A. polyphaga amoeba. (d) Percentage of A. polyphaga amoebae with adhered/internalized bacteria. FL3, red fluorescence signal; MFI, mean fluorescence intensity; ns, not significant. *, P ⬎ 0.05 versus pH 7; ***, P ⬍ 0.001 versus pH 7.

29). Here we show for the first time that coincubation with Acanthamoeba affects C. jejuni survival in acidic environments. All three of the Acanthamoeba species tested (A. polyphaga, A. castellanii, and A. rhysodes) prolonged the survival of C. jejuni at low pH compared to that in the absence of amoebae. It was previously shown that C. jejuni can obtain protection from free chlorine residues by internalization into amoebae or ciliates compared to pure bacterial cultures (17). Collectively, these observations indicate that C. jejuni organisms associated with

TABLE 1. Swarm diameters of C. jejuni cells pretreated with PBS at different pHs for 1 h before inoculation onto soft agar plates at pH 7a pH

Swarm diameter (cm)

P value

7 5 4 3 2

1.5 ⫾ 0.03 1.9 ⫾ 0.03 1.4 ⫾ 0.02 1.2 ⫾ 0.03 0

⬍0.0001 0.047 ⬍0.0001

a Data are expressed as means ⫾ SEM for three independent observations with six replicates each. P values are for comparison to data for pH 7.

amoebae, either as internalized cells or as cells adhered to the amoeba cell wall, may acquire protection against hostile environmental factors. In this study, we compared the survival of C. jejuni cells coincubated with A. polyphaga to that in the absence of amoebae. The coincubations were set up in two different ways: either the bacteria were added to amoebae before acidification of the medium, or they were added after acidification. At pH 4 and pH 5, there were remarkable differences between the two treatments. When C. jejuni cells were added to the amoebae after the pH had been adjusted, bacterial survival was significantly higher than when they were added before the pH adjustment or than that of the controls. This indicated a more efficient internalization of C. jejuni into amoebae when bacteria were added after acidification of the medium. Therefore, we hypothesized that an acidic environment might trigger internalization of C. jejuni into A. polyphaga. Indeed, both flow cytometric analysis and microscopic determination of the extent of bacterial adhesion/internalization into A. polyphaga showed that the numbers of adhered/internalized C. jejuni cells were considerably higher at pH 4 and 5 than at pH 7. The

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number of amoebae that had taken up bacteria also increased at pH 4 and 5 compared to that at pH 7. Interestingly, it has been demonstrated that H. pylori, which is taxonomically closely related to C. jejuni, can actively move away from the low pH of the ventricular lumen into the mucosal layer, where the pH is higher (8). By motility assay on soft agar plates, we found that the motility of C. jejuni decreased with decreasing pH. However, when bacteria were pretreated at pH 5 for 1 h before addition to soft agar plates with neutral pH, there was an increase in bacterial motility, suggesting that a transient exposure to a low pH might trigger C. jejuni motility. Other studies have found that C. jejuni can show a tolerance response after brief exposure to mild acid stress (18, 22). Collectively, these results indicate that in a moderately acidic milieu (pH 4 to 5), C. jejuni can actively migrate toward Acanthamoeba trophozoites, and interactions with amoebae are increased either by an active bacterial invasion mechanism or as a result of an increased density of bacteria at the amoeba surface. At pH 1 to 2, on the other hand, C. jejuni survived only for a short time, even when coincubated with amoebae. Flow cytometric data as well as microscopic quantification of adhered/internalized C. jejuni cells at pH 2 and pH 3 showed that neither the number of adhered/internalized bacteria per amoeba nor the number of trophozoites associated with bacteria was significantly increased compared to that for incubation at pH 7. A previous study has shown that C. jejuni demonstrates signs of degenerative stress already at pH 4 (27), possibly caused by accumulation of H⫹ in the cytoplasm (12). It has also been shown that H. pylori cells lose their motility within a few minutes after exposure to pH 4 (26). In a swarming assay on soft agar plates with neutral pH, we found that C. jejuni cells pretreated for 1 h at pH 4 and below showed reduced motility compared to bacteria pretreated at pH 7. These results suggest that at very low pH, C. jejuni cells are unable to escape the unfavorable surroundings due to impaired motility. Instead, they will succumb in the acid, while the bacteria that are already inside the amoebae will be protected to some extent. In summary, our data suggest that at moderately low pH (pH 4 to 5), C. jejuni may be able to migrate away from an acidic environment toward a protective host or toward the higher pH of the ventricular mucosa until the luminal pH is increased, e.g., by food intake. C. jejuni might even be residing inside an amoeba when it is ingested, which could provide a mechanical barrier against the harsh conditions during gastric passage. At very low pH, planktonic C. jejuni cells are instead killed, while those associated with amoebae seem to be protected for some time. The results might have important implications for the role of amoebae in C. jejuni epidemiology, since C. jejuni organisms share many environmental locations with protozoa (14, 15). Contamination of industrial poultry with Campylobacter spp. is a huge problem, and poultry is the main source of Campylobacter sp. transmission to humans. As in every other wet environment, amoebae are also common inhabitants of poultry plants. A recent study identified no fewer than 91 morphotaxa and 22 unique phylotypes of unicellular eukaryotic organisms, almost half of which were amoeboid species, in three different poultry farms surveyed (3). Another study showed that chickens can be colonized with C. jejuni internalized in A. castellanii, and the authors concluded that the amoebae might facilitate

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colonization (30). One strategy for disinfection of poultry farms after Campylobacter sp. contamination is by washing of the interior of buildings with mild acid. Acanthamoeba spp. have been shown to survive and grow at a pH range of 4 to 12 (16). Our observations suggest that Acanthamoeba spp. can tolerate transient changes in pH, at least down to pH 3, and can support intracellular survival of C. jejuni in acidic environments. Furthermore, our findings suggest that mild acid treatment of poultry stables might even trigger internalization of C. jejuni into amoebae, and hence such treatment could have an effect that is contrary to the intended result. This is supported by previous work showing that acidification can sometimes increase the risk for colonization (21). Although this has been poorly studied in the field, the results imply that disinfection methods targeting amoebae might result in better eradication of Campylobacter spp. and warrant further studies. ACKNOWLEDGMENTS This work was supported financially by the Royal Swedish Academy of Agriculture and Forestry (KSLA-H-482), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS 2007-438), the Health Research Council of Southeast Sweden (FORSS), and Sparbankstiftelsen Kronan. We thank Gunnar Gunnarsson for valuable comments regarding the statistical analysis. REFERENCES 1. Axelsson-Olsson, D., P. Ellstrom, J. Waldenstrom, P. D. Haemig, L. Brudin, and B. Olsen. 2007. Acanthamoeba-Campylobacter coculture as a novel method for enrichment of Campylobacter species. Appl. Environ. Microbiol. 73:6864–6869. 2. Axelsson-Olsson, D., J. Waldenstrom, T. Broman, B. Olsen, and M. Holmberg. 2005. Protozoan Acanthamoeba polyphaga as a potential reservoir for Campylobacter jejuni. Appl. Environ. Microbiol. 71:987–992. 3. Bare, J., K. Sabbe, J. Van Wichelen, I. van Gremberghe, S. D’Hondt, and K. Houf. 2009. Diversity and habitat specificity of free-living protozoa in commercial poultry houses. Appl. Environ. Microbiol. 75:1417–1426. 4. Black, R. E., M. M. Levine, M. L. Clements, T. P. Hughes, and M. J. Blaser. 1988. Experimental Campylobacter jejuni infection in humans. J. Infect. Dis. 157:472–479. 5. Blaser, M. J., H. L. Hardesty, B. Powers, and W. L. Wang. 1980. Survival of Campylobacter fetus subsp. jejuni in biological milieus. J. Clin. Microbiol. 11:309–313. 6. Blaser, M. J., J. G. Wells, R. A. Feldman, R. A. Pollard, and J. R. Allen. 1983. Campylobacter enteritis in the United States. A multicenter study. Ann. Intern. Med. 98:360–365. 7. Cawthraw, S. A., T. M. Wassenaar, R. Ayling, and D. G. Newell. 1996. Increased colonization potential of Campylobacter jejuni strain 81116 after passage through chickens and its implication on the rate of transmission within flocks. Epidemiol. Infect. 117:213–215. 8. Croxen, M. A., G. Sisson, R. Melano, and P. S. Hoffman. 2006. The Helicobacter pylori chemotaxis receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa. J. Bacteriol. 188:2656–2665. 9. Doyle, M. P., and D. J. Roman. 1982. Response of Campylobacter jejuni to sodium chloride. Appl. Environ. Microbiol. 43:561–565. 10. Greub, G., and D. Raoult. 2002. Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study. Appl. Environ. Microbiol. 68:3076–3084. 11. Hansson, I., I. Vagsholm, L. Svensson, and E. Olsson Engvall. 2007. Correlations between Campylobacter spp. prevalence in the environment and broiler flocks. J. Appl. Microbiol. 103:640–649. 12. Hill, C., B. O’Driscoll, and I. Booth. 1995. Acid adaptation and food poisoning microorganisms. Int. J. Food Microbiol. 28:245–254. 13. Humphrey, T., S. O’Brien, and M. Madsen. 2007. Campylobacters as zoonotic pathogens: a food production perspective. Int. J. Food Microbiol. 117:237–257. 14. Huws, S. A., A. J. McBain, and P. Gilbert. 2005. Protozoan grazing and its impact upon population dynamics in biofilm communities. J. Appl. Microbiol. 98:238–244. 15. Jones, K. 2001. Campylobacters in water, sewage and the environment. Symp. Ser. Soc. Appl. Microbiol. 2001:68S–79S. 16. Khan, N. A. 2006. Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol. Rev. 30:564–595.

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