Survival capability of Campylobacter upsaliensis under environmental stresses

Campylobacter upsaliensis has been recognized as an emerging pathogen. However, little is known about its survival in the environment. To evaluate its survival capability, we estimated the reduction in viable counts of C. upsaliensis after aerobic exposure to starvation in phosphate-buffered saline (PBS), acidity (pH = 4.3), high osmolarity (4% NaCl), and dryness in wet pulp disks at different temperatures. Also, survival in dog feces and dog food at variable temperate was assessed. Campylobacter upsaliensis remained culturable under starvation for 4 days at 25 °C and for 10 weeks at 4 °C. C. upsaliensis was also recoverable after exposure to high osmolality for 9 days, dryness for 5 days, and acidity for 2 days, respectively. Similarly, C. upsaliensis survived in dog feces and dog food for several days at 25 °C and weeks at 4 °C. The survival capability of the organism was dependent on the water content, and also temperature. Notably, the tested C. upsaliensis strain was less resilient under all tested conditions than a C. jejuni strain used as a control. The findings showed that C. upsaliensis is able to survive under various environmental stresses, suggesting that it could pose a potential threat to public health.


Introduction
Campylobacter upsaliensis (Cups), the most common Campylobacter species found particularly in dogs, has been recognized as an emerging human pathogen [1]. The Cups infection is associated with a self-limiting diarrheal disease in most human cases; other serious conditions have also been reported, including bloody diarrhea, bacteremia, Guillain-Barré syndrome and hemolyticuremic syndrome [2,3]. Epidemiological studies have indicated that not only indirect transmission such as foodborne infection but also direct contact with infected dogs poses a significant risk for Cups infection in humans [3]. Children are thought to be more vulnerable to this risk. In fact, a significant association between cases of the infection in the ≤ 5-year age group and the presence of a puppy in the household has been demonstrated [4,5].
Although Campylobacter jejuni (Cj) requires microaerophilic conditions, this pathogen has acquired the ability to adapt to severe environmental conditions to maintain its life cycle [6]. However, little is known about the survival capabilities of Cups in the environment.
The present study was performed to evaluate the survival of Cups under various environmental stresses. We examined the stress responses of Cups to starvation, dryness, acidity, and osmolality in an aerobic atmosphere at 4 °C and 25 °C. We also examined its survival in dog feces and dog food at both ambient temperatures.

Survival of Campylobacter species exposed to starvation, dryness, acidity and high osmolality
Survival to starvation The 48-h cultivated Cups and Cj cells on blood agar plates were harvested in 10 mM phosphate-buffered saline (PBS, pH 7.2) and the optical density at 550 nm (OD 550 ) was adjusted to 0.1, representing 8 log 10 colony-forming unit (CFU)/ml. Aliquots of bacterial suspension (1 ml) were dispensed into glass tubes and covered with an aluminum cap. They were cultured aerobically in incubators at 25 °C or 4 °C. After predetermined incubation periods, the number of CFU in the PBS was measured by direct plating on blood agar.
Survival to dryness Aliquots of 50 μl of bacterial suspension (0.1 OD 550 ) were dropped onto sterilized pulp disks 10 mm in diameter used for detection of antimicrobial agents (Toyo Roshi, Tokyo, Japan). These were placed in covered petri dishes 90 mm in diameter (Iwaki, Tokyo, Japan) and incubated aerobically at 25 °C or 4 °C. After predetermined incubation periods, the weight variation of the pulp disks was measured, and then the number of CFU in the disk was determined by direct plating on blood agar. Survival to acidic or high osmolality stress was conducted as described elsewhere [7], with some modifications. In brief, the pH of Brucella broth (Beckton Dickinson, MD, USA) was adjusted to 4.3 by adding hydrochloric acid (HCl) (Nacalai Tesque, Kyoto, Japan) and used as a form of acidic stress. For the high osmolality stress test, 3.5 g of sodium chloride (NaCl) (Nacalai Tesque) was added to 100 ml of Brucella broth resulting in a final concentration of 4% NaCl, since Brucella broth already contains 0.5% NaCl. The 48-h cultivated Campylobacter spp. were inoculated in the modified Brucella broths, and the OD 550 of both inoculated broths was adjusted to 0.1. The bacterial counts were conducted as described above.

Bacterial survival in dog feces, and dog food artificially spiked with Campylobacter spp.
Survival in dog feces Stool material was collected from a healthy adult dog and confirmed to be Campylobacter spp.-negative using enrichment followed by direct plating culture [8]. This fecal material (20 g) was then inoculated by mixing 20 ml of 8 log 10 CFU/ml Cups and Cj in a sterile stomacher bag (Central Scientific Commerce Inc., Tokyo, Japan), and homogenized for 2 min. Then, 1-g aliquots of feces were placed in covered petri dishes 35 mm in diameter (Iwaki) and incubated aerobically at 25 °C or 4 °C. After predetermined incubation periods, the number of culturable cells in the fecal sample was measured by direct plating on Skirrow selective agar plates (Kanto Kagaku) after 4 days of incubation at 37 °C under microaerobic conditions as described above.
Survival in dog food A wet-type dog food (water content 80%) and a dry-type dog food (water content 10%) whose major ingredients were chicken meat and beef were purchased from a pet shop. Each food (20 g) was inoculated with 20 ml of 8 log 10 CFU/ml bacterial cells in a sterile stomacher bag. The wet-type food was then homogenized for 2 min but the dry type was suspended for 10 min before homogenization. The incubation, the food weight measurement, and the CFU count were conducted as described above.

Statistical analysis
Each experiment was repeated three times and mean microbial counts were converted to log 10 CFU/g. Pearson's correlation coefficient (R) was used to examine the relation between Cups and Cj counts and the respective weights of feces, food and wet pulps. Statistical significance was defined as P ≤ 0.05.

Survival of campylobacters in PBS under an aerobic atmosphere
The viable count of Cups fell below the detection limit after 5 days of incubation at 25 °C, while Cj did so at 6 days (Fig. 1A). In contrast, the culturabilities after incubation at 4 °C extended until 9 weeks for Cups and until 10 weeks for Cj (Fig. 1B). An environment contaminated with Campylobacter, particularly water, can pose a possible risk for transmission to animals and humans [9] and the present study showed that Cups retained its culturability in PBS under starvation stress and an aerobic atmosphere for several weeks at 4 °C. Indeed, in a case study of Cups infection in a hiker, the source of infection was suspected to be drinking of unsterilized spring water [10].

Survival of campylobacters under acidity, osmotic stress, and dryness
Cups showed a marked decline in the viable counts (~ 6 log 10 ) after 2 days of incubation in 4.3 pH medium at 4 °C and was not detectable by 3 days. Cj was less severely affected and was culturable until 3 days of incubation (Fig. 1C). Cj was reported to grow well at pH 6.5-7.5, but its survival rapidly diminished at acidic pH [11]. The high sensitivity of Cups to acidity, suggesting lower survival under gastric acidity, may account for the lower incidence of Cups infection in humans. However, it has been suggested that ingestion of pathogenic Campylobacter with water [9] or certain foods [12], which act as buffers, may increase their survival in extremely acid conditions. For osmolality stress, Cups survived for 9 days in highosmolarity broth medium (4% NaCl) at 4 °C, but was more vulnerable than Cj (Fig. 1D). Under the same incubation conditions, Cj was culturable for 10 days with a ~ 3 log 10 decline from the initial count. This agreed with a previous study that demonstrated a decrease of about 3 log 10 in the Cj count within 14 days at 4 °C in the presence of 4.5% NaCl [13].
For dryness stress, the counts of both species declined simultaneously with continuous loss of moisture content in the inoculated pulp disks at 4 °C. Cups and Cj showed high sensitivity to dryness, and their viable counts fell below the detection limit by 6 and 9 days at 4 °C, respectively (Fig. 1E); this highlighted the importance of moisture content for survival of both species in the environment. This is in accord with previous reports that demonstrated long survival of Cj for more than 80 days in filter-sterilized stream water at low temperature [14] and a rapid decline in numbers under dry conditions [15]. Notably, both species failed to survive dryness, acidity and high osmolarity at 25 °C for 24 h, which highlight the key role of ambient temperature in survival of Campylobacter spp. to stresses in low nutrient environment.

Survival of Campylobacter species in dog feces
Cups survived in dog feces for about 3 days at 25 °C and for a longer time at low temperature ( Fig. 2A, B). A marked difference in the survival of Cups relative to Cj was notable at the 1st week at 4 °C (~ 3 log 10 ), suggesting that temperature may not be the sole factor influencing the survival of Cups in dog feces. Notably, the moisture content of the feces affected the survival of both of the well as that of other enteric bacteria such as Salmonella and Escherichia coli [16]. Previous reports have suggested that Cj survives for a variable time in cattle and poultry feces [17,18]. However, there have been no available data on survival of Campylobacter spp. in dog feces.

Survival of Campylobacter species in dog food
In this study, we examined the survival of Cups and Cj in wet-and dry-type dog foods incubated aerobically at 25 °C and 4 °C. The viable count of Cups fell below the detection limit at 4 days after incubation at 25 °C (Fig. 2C) and at 2 weeks at 4 °C (Fig. 2D), while Cj was still culturable after 5 days at 25 °C (Fig. 2C) and after 5 weeks at 4 °C (Fig. 2D). The culturability of both Campylobacter spp. was shorter in the dried food than in the wet food (Fig. 2E, F). The survival of Campylobacter spp. in dog food (Fig. 2C-F), was correlated with its moisture content at both temperatures (R = 0.7-0.9, P ≤ 0.01). Many factors affect the survival of microorganisms in foods, including moisture, osmolarity and acidity [19]. The relatively short survival of Cups in dog food, especially dry-type food, may be attributed to sensitivity to these stressors. The present data on the survival of Cups in dog feces and dog food suggest an infectious source of transmission to other animals and to humans, although epidemiological data for dogs have been unavailable up to now.
Campylobacter spp. survive in the environment through mechanisms such as aerotolerance, biofilm formation, adaptive tolerance responses and transformation to a viable but nonculturable (VBNC) [6]. In all of the present experiments, the culturability of Cups was lower than that of Cj to a varying degree. This inter-species difference in survival tolerance to stress has been reported before [20]. One possible explanation for this is the variation in catalase production by these organisms. It is considered that catalase produced by Campylobacter spp. protects them from damage caused by oxygen radicals [21]. As Cups was originally described as a catalase-negative or weakly positive organism [22], this may account for its lower survival capability in comparison to Cj under aerobic conditions.
The risk arising through continuous and daily contact with household dogs, especially for children, has been emphasized. For prevention of new human cases, there is an urgent need for better public awareness and improved detection techniques for Cups. The present study provides the first information on the survival of Cups in the rearing environment of dogs, which may help to clarify possible routes of infection and prevent transmission of this pathogen to humans.

Limitations
This study has some limitations. We have investigated only one type strain as a representative Campylobacter spp. and only two temperatures as an initial proof of concept. Furthermore, it is suggested that the phenotypic differences may be attributed to the high genetic diversity among Campylobacter spp. Therefore, the differences in survival patterns between Cups and Cj may be reflected in part by interspecies differences in stress response genes. Although the whole genomes of some Cups strains have been determined, the key genes related to the survival strategies of Cj have not yet been fully examined. While this study limited our ability to assume causality, findings may inform the direction of future research to understand the survival abilities of campylobacters under environmental stresses.