Campylobacter jejuni is one of the main bacterial causes of gastroenteritis in Europe, with about 300,000 reported cases per year (EFSA, 2021). The infection by this pathogen usually leads to the development of gastroenteritis (campylobacteriosis) with fever, abdominal pain, dysentery, and nausea. In most cases, the illness is self-limiting with a duration varying from a few days to weeks (Horrocks et al., 2009; Silva et al., 2011). In some cases, such as in immunocompromised people, C. jejuni can be a precursor of autoimmune diseases, such as Guillain-Barré syndrome, reactive arthritis, irritable bowel syndrome, and inflammatory bowel disease (Rees et al., 1995; Ang et al., 2001; Ternhag et al., 2008). As the main reservoir of C. jejuni is the gastrointestinal tract of wild and domestic animals, especially birds, campylobacteriosis is mainly associated with the consumption of raw or undercooked poultry products (Kaakoush et al., 2015; Skarp et al., 2016). However, more and more data have been published showing that non-food sources, such as drinking and surface water, can participate in the dissemination of the disease (O’Reilly et al., 2007; Uhlmann et al., 2009; Whiley et al., 2013; Guzman-Herrador et al., 2015; Moreira and Bondelind, 2017; Ahmed et al., 2020; Hyllestad et al., 2020; Kuhn et al., 2020; Tzani et al., 2020). C. jejuni cells were also isolated from rivers (Khan et al., 2014), agricultural waters (Mulder et al., 2020), lakes (Kemp et al., 2005), children’s pool waters (Sawabe et al., 2016), beach sand samples (Yamahara et al., 2012) and playgrounds (French et al., 2009). Fecally contaminated drinking water has been associated with several Campylobacter outbreaks in Sweden (Martin et al., 2006), Finland (Miettinen et al., 2001; Hänninen et al., 2003), England (Smith et al., 2006), the United States (Pedati et al., 2019), Norway (Paruch et al., 2020), New Zealand (Bartholomew et al., 2014; Gilpin et al., 2020) and Denmark (Kuhn et al., 2017). Most of the outbreaks have been attributed to the consumption of untreated groundwater.

Campylobacter jejuni is unique among foodborne pathogens in its obligate microaerophilic nature and narrow range of growth temperatures, with a minimum growth temperature ranging from 31 to 36°C (Hazeleger et al., 1998; Davis and DiRita, 2008), and optimum at 42°C. Some studies suggest that despite its strict growth requirements, C. jejuni has the ability to survive under conditions that otherwise inhibit its growth, such as an atmospheric concentration of oxygen. Exposure to an aerobic atmosphere leads to oxidative stress caused by the formation of reactive oxygen species (ROS), which among other things destroy numerous Fe-S proteins of C. jejuni. Although a limited number of ROS detoxification enzymes are expressed in C. jejuni (Atack and Kelly, 2009), a few previously isolated isolates were reported to be aerotolerant (Oh et al., 2015a; Bronnec et al., 2016; Karki et al., 2018). They can effectively survive under aerobic conditions (AC) and therefore pose a serious risk of food contamination and human infection. The hyper-aerotolerant isolates (those remaining viable after 24 h of aerobic incubation) of C. jejuni were so far isolated from raw poultry meat, a duck slaughterhouse, human clinical samples, and dairy products (Rodrigues et al., 2015; Guk et al., 2019; Kiatsomphob et al., 2019; Mouftah et al., 2021). Several studies suggested that C. jejuni also has the potential to survive under non-permissive temperature conditions. It is known that cells of C. jejuni exposed to ambient temperature remained viable for a few days, while cells cultivated at 4°C survived for several weeks (Terzieva and McFeters, 1991; Buswell et al., 1998), or even months when transformed into a non-cultivable coccoid form (Rollins and Colwell, 1986; Jones et al., 1991; Hazeleger et al., 1995).

The survival of Campylobacter in water is influenced by many factors, such as the concentration of dissolved oxygen (Buswell et al., 1998), presence of ammonium, chloride ions and phosphate (Strakova et al., 2021), and presence of other microorganisms. According to several recent studies, the survival of C. jejuni can be enhanced in the presence of Acanthamoeba spp., which is an amoeba frequently found in water and soil (Siddiqui and Khan, 2012). It was previously shown that bacterial interactions with Acanthamoeba are a defense mechanism of various pathogens, e.g., Legionella pneumophila, Vibrio spp., Pseudomonas aeruginosa, Listeria monocytogenes, Helicobacter pylori, Escherichia coli and Mycobacterium avium, and may even act as a vector for transmission of these pathogens to hosts (Rowbotham, 1980; Cirillo et al., 1999; Winiecka-Krusnell et al., 2002; Reyes-Batlle et al., 2017). As for C. jejuni, cocultivation with the amoeba resulted in a delayed decline of bacterial numbers and a longer survival period at both sub-optimal temperatures and under AC (Axelsson-Olsson et al., 2005, 2010a,b; Baré et al., 2010; Bui et al., 2012a; Griekspoor et al., 2013).

It was previously reported that co-incubation with Acanthamoeba resuscitates C. jejuni cultures (Axelsson-Olsson et al., 2005, 2007; Reyes-Batlle et al., 2017), provides C. jejuni with increased acid resistance (Axelsson-Olsson et al., 2010a,b), protects it from chlorine and other chemical disinfectants (King et al., 1988; Snelling et al., 2005), and improves the survival of C. jejuni in milk and orange juice (Olofsson et al., 2015). However, the molecular mechanisms of interactions between C. jejuni and amoebas are not well understood, and the results of related publications are often conflicting. Some studies suggest C. jejuni adheres to the surface of the amoeba and survives extracellularly (Baré et al., 2010; Bui et al., 2012b), while others claim that Campylobacter cells enter the intracellular space of the amoeba, where it is able to survive and potentially multiply (Axelsson-Olsson et al., 2005, 2007, 2010b; Snelling et al., 2005; Griekspoor et al., 2013; Olofsson et al., 2013). The discrepancy could be partially explained by using various collection strains of C. jejuni and different strains of amoebae, which makes the results difficult to compare.

The increased aerotolerance of various C. jejuni isolates together with the prolonged survival of cells due to low temperatures and interactions with amoebas suggest that previously overlooked environmental niches might contribute to the transmission of C. jejuni and subsequently to the numbers of human infections. However, the majority of Campylobacter-related research solely uses isolates from food and clinical samples, whose phenotypic features might differ from the environmental isolates continuously stressed by low nutrition, low temperature, and a high concentration of oxygen. To fill this knowledge gap, this work focused specifically on the survival of several water isolates in comparison with one isolate that originated from a clinical sample, and one from raw chicken meat.

All experiments were performed in three independent biological replicates. Viable counts were log₁₀ transformed for further analysis. Calculations and graphs were processed with Microsoft Excel 2016.

As was mentioned above, water is an important source of human Campylobacter infections. An enhanced ability to survive in this low-nutrient, low-temperature, aerobic environment could be an important property, especially among isolates involved in water-related outbreaks. It can be expected that since it passes through various environments, C. jejuni overcomes harsh conditions, especially relatively high levels of oxygen and temperature swings, which can directly affect its viability. In this study, 14 isolates of C. jejuni were selected for further analyses, 12 of them being originally isolated from surface water in the Czechia. The general resilience of the isolates was evaluated by their antibiotic resistance, ability to survive and potentially grow under an aerobic atmosphere and at low temperature, and by their interactions with Acanthamoeba polyphaga. Recently, an increase in the resistance of Campylobacter spp. to antibiotics has been reported in several countries, including the Czechia (Serichantalergs et al., 2007; Bardon et al., 2009). Factors contributing to this increase could include the veterinary use of antibiotics as prophylaxis or for the treatment of animal diseases (Skandalis et al., 2021). In our study, more than half of the isolates (64.3%, 9/14) were resistant to at least one of the tested antibiotics, usually CIP. Interestingly, TET resistance was only observed in the isolates from wastewater treatment plants and from the clinical sample. This suggests that TET resistance was either acquired by the isolates in the wastewater treatment plant through horizontal gene transfer, or they originate from clinical cases and later reached the plant through the sewage systems. It is known that wastewater and sewage systems are a major reservoir of resistant bacterial populations due to the collection of antibiotic waste from humans and animals, improper disposal of drugs, and from factories, hospitals, and veterinary clinics (Nadimpalli et al., 2020). The resistance profiles of the waterborne C. jejuni suggest that these isolates may pose a significant public health threat, as quinolones (including CIP) are commonly used for campylobacteriosis treatment in clinical cases, especially the severe ones (Guk et al., 2019). In addition, there is evidence that patients infected with antimicrobial-resistant Campylobacter species have longer-lasting diarrhea than patients infected with antimicrobial-susceptible isolates (Nelson et al., 2004).

Despite the general perception that C. jejuni is microaerobic and therefore sensitive to oxygen, our data showed that C. jejuni isolates are able to survive and even multiply under AC. Some studies have already reported on the ability of C. jejuni isolates to survive in the presence of oxygen (Kaakoush et al., 2007; Oh et al., 2015a,b, 2017). However, those studies were mostly focused on the simple survival of the isolates in AC, not on their active growth. Recently, Rodrigues et al. (2015) characterized a unique human isolate of the C. jejuni strain, named Bf, that can grow under AC, suggesting that some isolates are highly resistant to aerobic stress. According to our results, the ability of aerobic growth is more common than expected, as all our waterborne isolates were able to adapt, develop aerotolerance and grow aerobically. Some of them were even able to maintain this ability even after short-term storage in the deep freezer. The microorganisms, once adapted, behaved as conventional aerobes and could be continuously maintained in aerobic subcultures for up to 3 days. In most isolates, aerobic growth was weak during the first 24 h, but improved with each subcultivation on fresh Karmali plates. It seems that aerotolerance is not a matter of different genetic background of the isolates, since the results of three independent direct cultivations under an aerobic atmosphere gave different results. Bronnec et al. (2016) have already sequenced one aerobic isolate of C. jejuni, but according to their results, the C. jejuni Bf gene repertoire required for resistance to oxidative stress did not differ from other isolates. As the aerotolerance was induced in all isolates, but was not retained for a long period of time, it is instead a matter of the regulation of gene expression. However, further research is needed to explain the regulatory mechanisms participating in the development of this aerotolerance.

The sensitivity to the concentration of oxygen goes hand in hand with the cultivation temperature. In many studies, it has already been mentioned that low temperature is an important factor affecting the survival of C. jejuni in an aqueous environment, including surface waters and sterilized water (Terzieva and McFeters, 1991; Buswell et al., 1998; Thomas et al., 1999; Tatchou-Nyamsi-König et al., 2007; Bronowski et al., 2014). To test whether the waterborne isolates are better adjusted to low temperatures than other isolates, they were cultivated at 7°C, both under aerobic and microaerobic conditions. All isolates were indeed able to survive at low temperature, which is consistent with previous observations that C. jejuni survives well under unacceptable temperatures for growth (Chynoweth et al., 1998; Chan et al., 2001; Bhaduri and Cottrell, 2004; Cole et al., 2004). However, their survival was more prolonged under a MAC than under air. All 14 isolates maintained their viability up to 4 weeks of microaerobic incubation without any strong decline in cell numbers, while under AC, the cultivability lasted for 1–3 weeks. In aqueous environments, the concentration of dissolved oxygen significantly varies between the different types of surface waters, as it is decreased by high pollution with organic substances, low flow rate, increased temperature, and even the presence of high numbers of competing microorganisms (Terzieva and McFeters, 1991). Thus, at least part of the aquatic environment could in fact be microaerobic. One of the studies demonstrated that C. jejuni populations survived longer in stationary than aerated water. Moreover, an increase in the cultivation temperature led to a decrease in the possibility of extracting viable C. jejuni (Rollins and Colwell, 1986). Similarly, Chynoweth et al. (1998) observed that isolates cultivated under microaerobic conditions in sterile stream water at 5°C survived better than when cultured aerobically. However, Buswell et al. (1998) showed that the degree of oxygenation affected survival in an unpredictable manner, and the effect was especially noticeable at lower temperatures. The survival of some isolates improved at lower oxygen levels, while it decreased for others.

Unlike under laboratory conditions, environmental water is full of other organisms that can directly affect the survival of Campylobacter, such as various protozoa. Therefore, selected waterborne isolates were investigated for their ability to interact with and be protected by A. polyphaga, as the protective effect of cocultivation with the amoebae had been previously suggested in several studies (Axelsson-Olsson et al., 2005, Axelsson-Olsson et al., 2010b; Snelling et al., 2005; Baré et al., 2010). According to our results, the cocultivation of all isolates for 3 h resulted in the survival of about 40% of cells treated with an otherwise lethal dose of gentamicin. The question remains of whether the numbers of protected cells were given by the capacity of amoebas and their vacuoles, or by the fact that within the 3 h of co-incubation some C. jejuni cells remained attached to the surface of the amoeba, and didn’t have enough time to enter the protective vacuole. On the whole, our results are in accordance with the results of Axelsson-Olsson et al. (2005), who demonstrated a prolonged intracellular survival of motile C. jejuni cells within amoebae, and with Vieira et al. (2017), who reported that the collection strain 81–176 can invade and survive inside A. polyphaga at 25°C for a certain period of time. However, it appears that not all Acanthamoeba species have the same effect. Our results conflict with a previous study by Dirks and Quinlan (2014), who showed that C. jejuni survives the assay independently of the presence of A. castellanii, and that the internalization of C. jejuni by A. castellanii is not consistent. Baré et al. (2010) also showed that co-culture with A. castellanii did not increase the numbers of either bacteria or amoebae, but delayed the decline and prolonged the survival of Campylobacter.

Interestingly, during the examined stress conditions, the water isolates of C. jejuni were resistant and survived at the same rate as the clinical and the meat isolate. However, no connection was found between the origin of the isolates and their particular features, except for the TET resistance observed in isolates from wastewater treatment plants and the clinical sample. In conclusion, this study proved that waterborne isolates of C. jejuni are able to adapt to different stress conditions that should supposedly impair their survival in the environment. As the resilience of the water isolates was comparable to both clinical and raw meat isolates, we suggest that water should be regarded as a significant reservoir of C. jejuni, and isolates of various water origins should be included in further research of this emerging pathogen.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

ES and HM designed the experiments. ES performed the experiments and prepared the manuscript. KD and HM reviewed the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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