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Survival of stress exposed Campylobacter jejuni in the murine macrophage J774 cell line
International Journal of Food Microbiology 129 (2009) 68–73
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Survival of stress exposed Campylobacter jejuni in the murine macrophage J774 cell line Maja Šikić Pogačar a, Roberta Rubeša Mihaljević b, Anja Klančnik a, Gordana Brumini c, Maja Abram b, Sonja Smole Možina a,⁎ a b c
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia Department of Microbiology, Medical Faculty, University of Rijeka, Brace Branchetta 20, HR-51000 Rijeka, Croatia Department of Medical Informatics, Medical Faculty, University of Rijeka, Brace Branchetta 20, HR-51000 Rijeka, Croatia
a r t i c l e
i n f o
Article history: Received 29 February 2008 Received in revised form 27 October 2008 Accepted 9 November 2008 Keywords: Campylobacter jeuni Environmental stress Macrophage Survival
a b s t r a c t Although campylobacters are relatively fragile and sensitive to environmental stresses, Campylobacter jejuni has evolved mechanisms for survival in diverse environments, both inside and outside the host. Their survival properties and pathogenic potential were assessed after subjecting food and clinical C. jejuni isolates to different stress conditions. After exposure to starvation (5 h and 15 h of nutrient depletion), a temperature shock (3 min at 55 °C) or oxidative stress (5 h and 15 h of atmospheric oxygen) we studied the culturability, viability and capability of adhesion, internalization and survival within the in vitro cell culture model using J774 murine macrophages. Starvation severely impaired C. jejuni culturability, particularly after 15 h of nutrient depletion. The number of viable cells decreased by 30–40%. Starved bacterial cells also showed a lower capability of adhesion, internalization and survival within macrophages. Despite the reduced culturability and viability of the heat treated cells, C. jejuni efficiently adhered to, and entered murine macrophages. However, the number of heat treated cells started to decrease more quickly than non-stressed cells. Within 24 h post infection all the cells were killed. The bacterial mechanisms involved in inactivating toxic oxygen products may enhance bacterial persistence through increased binding, entry and survival of both oxidatively stressed C. jejuni isolates inside the macrophages. Oxygen exposure increased the internalization and intracellular survival, although the cells cannot remain viable for extended periods within murine macrophages. However, any prolongation of survival in macrophages may increase the probability of transmission of bacteria in the host organism and have further implications in the pathogenesis of campylobacteriosis. This indicates that environmental stress conditions may be involved. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Campylobacter jejuni is a gram negative, slender, curved, and highly motile rod-like bacterium. It has microaerophilic and thermophilic character with a requirement for reduced levels of oxygen and an optimal growth temperature of 42 °C, probably reflecting an adaptation to the avian gastrointestinal system. Healthy chickens carry these bacteria in their gut causing contamination of raw poultry meat, which is considered the major source of human food-borne campylobacteriosis (Park, 2002). C. jejuni and related campylobacters are relatively fragile and sensitive to environmental stresses like drying, heating, disinfectants, acidity and oxygen exposure. But, despite fastidious growth requirements and increased environmental sensitivity, C. jejuni is recognized as a leading cause of human gastroenteritis worldwide (Adak et al., 2005), indicating that it has developed some mechanisms for survival
⁎ Corresponding author. Tel.: +386 1 423 11 61; fax: +386 1 256 62 96. E-mail address: [email protected] (S. Smole Možina). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.11.010
in diverse environments, both inside and outside the host (Murphy et al., 2006; Young et al., 2007). Stress-responsive alternative sigma factors, including RpoS in gram-negative bacteria (Ibanez-Ruiz et al., 2000) and σB in gram-positive bacteria with a low proportion of guanine-cytosine content, contribute to regulation of large sets of genes (Petersohn et al., 2001). As reported for L. monocytogenes, exposure to sublethal stresses might enhance the organism's virulence, e.g., by activating the expression of σB-regulated virulence genes (Ferrierra et al., 2001; Kazmierczak et al., 2003). Exposure of L. monocytogenes to different environments may have additional effects on virulence-related characteristics (Garner et al., 2006). Campylobacters lack many of the adaptive responses to environmental stresses known to enable survival of other food-borne pathogens, including alternative sigma factor σB, that contributes to transcription of stress response and virulence genes (Park, 2002). In spite the high prevalence of C. jejuni in human bacterial gastroenteritis, the pathogenesis of infection is still poorly understood. Once inside the human digestive system, campylobacter colonizes and attacks the mucosa of the small and large intestine.
M. Šikić Pogačar et al. / International Journal of Food Microbiology 129 (2009) 68–73
Attachment to intestinal epithelial cells is an early and important step in the infectious process leading to bacterial internalization. Examination of intestinal biopsies of humans (Chang and Miller, 2006), in vivo studies in animal models (McOrist et al., 1989; Russell et al., 1993; Vučković et al., 1998), together with in vitro experiments using cultured intestinal epithelial cells (Friis et al., 2005; Kalischuk et al., 2007; Mihaljević et al., 2007) have clearly demonstrated that C. jejuni can invade non-phagocytic intestinal epithelial cells. Internalization of C. jejuni is reduced at low temperature (Konkel et al., 1992), as well as upon exposure to heat shock or starvation, but seems to be increased when pre-cultivated under aerobic conditions for 5 h (Mihaljević et al., 2007). Once within epithelial cells, C. jejuni can survive (Konkel et al., 1992; Watson and Galán, 2008) and multiply intracellularly (Mihaljević et al., 2007). Thereafter, C. jejuni can gain access to underlying tissues and reach different cellular receptors and professional phagocytic cells. It is known that survival of bacteria within phagocytic cells represents a strategy to evade the host defences and allows proliferation and dissemination of bacteria throughout the host. On the other hand, once inside the macrophage, bacteria must be able to overcome a variety of unfavourable conditions triggered by the host's killing mechanisms, such as oxidative products, nutrient limitation, acidic pH and other stimuli. There are contradictory reports regarding the ability of C. jejuni to survive within macrophages, depending on the macrophage cell type and C. jejuni strain. There is evidence that viable campylobacters can be recovered from human monocyte cultures after extended periods (Hickey et al., 2005; Kiehlbauch et al., 1985) and that catalase-producing strains have an enhanced chance of intramacrophage survival (Day et al., 2000). On the contrary, other data indicate that intraphagocytic survival is not a
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common phenomenon during campylobacter infection because human derived monocytes/macrophages kill phagocytosed bacteria within 24 to 48 h (Wassenaar et al., 1997). It has been found that C. jejuni is targeted on lysosomes and therefore cannot survive inside mouse primary bone marrow-derived macrophages (Watson and Galán, 2008). The aim of our study was to analyse whether submission to an environmental stress may modulate the virulence characteristics of Campylobacter and give it an advantage in surviving in macrophages. We initially examined the changes of culturability and viability of C. jejuni in response to temperature shock, starvation, and atmospheric oxygen pre-treatment. After stress exposure, in an in vitro cell culture model using J774 murine macrophages, changes in adhesion, internalization and intracellular survival of two C. jejuni isolates, one clinical and one from food, were studied. 2. Materials and methods 2.1. Bacterial strains, culture media and growth conditions A food (poultry meat) isolate (K49/4) and a clinical isolate obtained from a patient with severe diarrhea were identified by phenotypic and molecular techniques as described previously (Zorman and Smole Možina, 2002). The strains were stored at −80 °C in a brain heart infusion broth supplemented with blood and glycerol, and subcultured prior to subjecting them to experimental procedures. Microaerobical (O2 5%, CO2 10%, and N2 85%) growth in Preston broth at 42 °C for 9 h induced the entry of the culture into the exponential growth phase, as determined previously (Klančnik et al., 2006).
Fig. 1. Culturability and viability of C. jejuni clinical (A) and food (B) isolates of non-stressed and stress exposed C. jejuni. The results are presented as relative rate ± SD (*P ≤ 0.05).
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2.2. Stress conditions Starvation, high temperature and oxidative stress were selected as bacterial stress conditions. For starvation, campylobacters were harvested by centrifugation (12,000 ×g at 4 °C for 5 min), washed, resuspended in Ringer solution (KH2PO4, 5 mM (Kemika), which was used as a multiple nutrient starvation medium, and incubated for 5 or 15 h microaerobically at 42 °C. To achieve heat shock the cells were shifted from 42 to 55 °C for 3 min and cooled on ice before analysis. For oxidative stress, the cells were exposed to atmospheric oxygen for 5 or 15 h. Non-stressed bacterial culture, taken at the same time as the stressed culture, served as the control. For culturability, viability, adhesion and invasion the relationship between stressed and nonstressed control cultures (relative rate) represented the influence of individual stresses. 2.3. Assessment of bacterial culturability and viability Culturability (CFU mL− 1) was assessed on Karmali agar plates (Oxoid, UK) after serial sample dilutions and microaerobic incubation at 42 °C for 24 or 48 h. Culturability experiments were independently repeated three or more times and the results expressed as log10 CFU/mL relatively to the non-treated control at the same time.
Total and viable counts were assessed using the LIVE/DEAD® BacLight™ viability kit (L-7012, Molecular Probes, Eugene, Oregon, USA) and microscopically examined (Eclipse TE300, Nikon, super high pressure mercury lamp power supply, X-60 oil immersion fluorescent objective, Nikon digital camera DXM 1200, Nikon Programing Equipment Laboratory Imaging Ltd., LUCIA 4.60, Tokyo, Japan) using blue excitation light as described previously (Klančnik et al., 2006). Viability is given as the percentage of viable cells relative to the total abundance obtained in 20 microscopic fields per filter. Viability experiments were independently repeated at least twice. The results are expressed as viable cells relatively to the non-treated control at the same time. 2.4. Adhesion, invasion and intracellular survival in J774 macrophages J774 macrophages were grown in RPMI 1640 medium (Institute of Immunology, Zagreb, Croatia) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 000 UI/mL penicillin and 10 000 μg/mL streptomycin (Gibco, London, UK). The cells were grown in tissue culture Petri dishes at 37 °C in a 5% CO2 humidified atmosphere. For all assays, 24-well tissue culture trays were seeded with approximately 5.0 × 105 macrophages per mL, and incubated for 24 h. The bacterial number was determined spectrophotometrically (absorbance mode at
Fig. 2. Adhesion and invasion ability of C. jejuni clinical (A) and food (B) isolates to J774 macrophages after stresses exposure. The results are presented as relative rate ± SD (*P ≤ 0.05).
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a wavelength of 600 nm). Infection was carried out by inoculating C. jejuni at approximate MOI of 100. Infected monolayers were incubated for 2 h to allow adhesion and invasion to occur. Then, the monolayers were washed three times with RPMI without an antibiotic to remove unbound bacteria. For the bacterial invasion assay, a medium containing 100 μg/mL of gentamicin was added for 1 h to kill extracellular bacteria. Following this period, monolayers were lysed with cold distilled water, and the released internalized bacteria were enumerated by plating serial dilutions on blood agar plates. For intracellular survival analysis, the invasion period was monitored 3, 5, 10, 15, 24 and 29 h post infection. The total number of bacteria associated with the macrophages (adherent + engulfed) was determined simultaneously by performing an invasion assay but without gentamicin treatment. The difference between the total and the number of intracellular bacteria was calculated and the number of adherent C. jejuni was obtained. Results for adhesion and invasion are expressed as relative rate ± SD of log10 CFU/mL of adherent or internalized bacteria, and for intracellular survival as the mean ± SD of log10 CFU/mL of internalized bacteria, for three independent measurements.
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2.5. Statistical analysis Data on bacterial counts from different experimental groups were compared using one-way ANOVA and Tukey-b tests for post-hoc analysis. All statistical values were considered significant at the P level of 0.05. Statistical analysis was performed using Statistica for Windows version 6.1 (Statsoft Inc., Tulsa, OK, USA). 3. Results 3.1. Culturability and viability of C. jejuni exposed to different stress conditions The culturability of clinical (A) and food (B) isolates of C. jejuni was monitored after exposure to temperature shock, atmospheric oxygen or nutrient deficiency (Fig. 1). After exposure to heat, a significant loss of culturability was observed for both isolates, although the food isolate showed slightly higher thermal resistance in comparison to the clinical strain. Starvation severely impaired bacterial culturability, particularly after 15 h of storage in a low nutrient medium. On the contrary, bacterial cultivation properties were unchanged following exposure to atmospheric oxygen. In parallel with the culturability analysis, viability changes of C. jejuni isolates were observed (Fig. 1). The level of viable cells decreased approximately 30–40% during heat shock and after starvation for 15 h. A decline of 20% was noticed in cell starved for 5 h; all oxidative stresses were milder. 3.2. Influence of environmental stresses on Campylobacter adhesion, invasion and intracellular survival
Fig. 3. Intracellular survival of control and stresses exposed C. jejuni, clinical (A) and food (B) isolates in J774 macrophage cell line. The bacterial number was determined by the CFU assay. Each point represents the log10 of the mean ± SD CFU/mL (*P ≤ 0.05).
Bacterial adhesion to, and uptake by J774 macrophages was assessed by plate-count assay after C. jejuni exposure to starvation, heat stress or atmospheric oxygen and compared to a non-stressed control. As shown in Fig. 2, there was no significant difference in the number of adhering or engulfed campylobacters after temperature shock in the case of both the clinical (A) and food (B), isolates. On the contrary, storage in a low nutrient medium, particularly for 15 h, significantly impaired bacterial adhesion as well as invasion properties. However, the food isolate was found to be more susceptible to starvation because a significant reduction in the number of adhering and ingested bacteria occurred already after 5 h of incubation in a low nutrient medium. Prolonged starvation for 15 h significantly reduced bacterial internalization. While 5 h exposure to atmospheric oxygen enhanced bacterial virulence properties according to the increased number of bound and intracellular bacteria, prolonged aeration (for 15 h) had no impact on the clinical strain but significantly decreased the adherence and invasion capability of the food isolate. To determine the role of selected stress conditions on C. jejuni survival within phagocytes in vitro, cultured murine J774 macrophages were infected with the clinical (Fig. 3A) and food (Fig. 3B) isolates and survival kinetics were analysed over a 48 h period. Both strains exhibited similar survival characteristics, undergoing a progressive decrease in the number of intracellular bacteria. It should be noted that 24 or 29 h post infection, no intracellular bacteria were recovered, indicating extensive killing by the macrophages. Pre-exposure of campylobacters to environmental stresses did not significantly influence the length of intramacrophage survival. However, considerable differences in the number of intracellular bacteria were observed. Bacterial starvation significantly decreased the survival period and the number of both C. jejuni isolates in J774 macrophages and correlated with the duration of pre-cultivation in the nutrient-poor medium. When the starvation period lasted for 15 h, C. jejuni was capable of surviving inside macrophages for only 10 h in the case of the food isolate, and 15 h in the case of the clinical isolate. Heat shock did not affect the initial phase of intracellular growth of C. jejuni, when the
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bacterial load was similar to the control level. However, the number of bacteria within macrophages started to decrease as early as 5 h post infection. Interestingly, our results confirmed that despite the microaerophilic nature of Campylobacter, pre-exposure to atmospheric oxygen enhanced its intramacrophage survival. When compared to the non-stressed control, viable clinical and food strains were recovered in higher numbers over the entire period of infection. On the other hand, prolonged oxidative stress (for 15 h), increased the sensitivity of the food strain to macrophage killing, since a lower CFU was detected, while the intracellular growth of the clinical isolate was not significantly affected. 4. Discussion Through the course of food borne infection food pathogens are subjected to host-imposed stress conditions, including the acidic stress of the stomach and then the oxidative stress of the host cell vacuole. Many intracellular food-borne bacteria capable of surviving in the intracellular compartment like Yersinia enterocolitica (Yamamoto et al., 1994) and Salmonella typhimurium (Ibanez-Ruiz et al., 2000) produce large amounts of stress proteins after phagocytosis. Their survival inside various cells, including phagocytes, has been proposed as a key element in the pathogenesis and manner of persistence in certain infections. Additionally, the mechanism used for defence against a single stress may provide bacteria cross-protection towards other unrelated hostile conditions. Bacterial cells can adapt to normally unfavourable growth conditions following a brief exposure to mild stress (Hill et al., 1995; Klančnik et al., 2008). It has been confirmed for Campylobacter spp. that submission to an initial stress may improve bacterial resistance to a second successive stress, enhancing their pathogenic characteristics (Murphy et al., 2003). Consequently, alterations in the growth environment of a bacterium may alter the expression of a variety of virulence factors (Biswas et al., 2007). The intracellular environment of a macrophage is one of the most hostile surroundings encountered by an invading bacterium. Therefore, the objective of this study was to examine in vitro in the cellculture model the efficiency of C. jejuni uptake into macrophages and to determine how long the bacteria can survive inside these cells. In addition, we were interested whether starvation, heat shock and atmospheric oxygen exposure induce changes in culturability/viability, as well as internalization and intramacrophage survival of clinical and food C. jejuni isolates. Starvation is the most significant factor in C. jejuni survival, coccoid cell formation and especially culturability (Klančnik et al., 2008). Also according to our results, starvation was the most powerful stress condition, which significantly affected C. jejuni culturability and viability, particularly after 15 h of nutrient depletion. This is consistent with the evidence that, unlike most other bacteria, C. jejuni does not exhibit increased survival during long periods of food deprivation, such as upon entry into the stationary phase (Kelly et al., 2001; Klančnik et al., 2006). When the effect of nutrient limitation on bacterial virulence was examined, starved bacterial cells of both strains showed a lower ability of adhesion, internalization and survival within macrophages. Previous studies confirmed that lack of nutrients and aging in Campylobacter induce disruption of both the inner and outer membrane structures and their functions (Kelly et al., 2001). The appearance of such sublethally injured cells, which prevail in the bacterial population, may be responsible for the significant loss of virulence and inhibition of bacterial growth. In its contamination cycle C. jejuni encounters a wide range of temperatures; during food processing, while entering the host and in the environment. Once exposed to 55 °C, a significant decrease of growth on agar, as well as in viability was detected. It is well known that immediately following heat shock C. jejuni increases the expression of a number of proteins (Parkhill et al., 2000). Many of
these proteins have been confirmed to play a role in bacterial–host interactions (Konkel et al., 1998) and could promote bacterial adherence and uptake in macrophages. However, contrary to expectation, C. jejuni efficiently adhered to, and entered murine macrophages, indicating that 3 min of heat shock was probably not enough for increased protein synthesis and that further incubation to monitor protein level changes, as suggested by Konkel et al. (1998), should be performed. The growth of heat treated bacteria appeared unaffected by the host intracellular milieu within the first 5 h post infection. However, afterwards both strains declined significantly in number and most of the intracellular bacteria were rapidly killed. Thus, the heat shock difference observed in Fig. 2 may simply be due to surface structural changes rather than increased protein production. Colonization of the host is a process involving adaptation of C. jejuni to different atmospheric oxygen concentrations. Although Campylobacters are generally considered microaerophilic, many reports indicate that C. jejuni has the ability of long-term aerobic adaptation (Murphy et al., 2003; Jones et al., 1993). Our results confirmed a similar outcome during exposure of campylobacter to oxidative stress. Microscopic counts of viable bacteria, as well as CFU numbers, exhibited no significant change after storage in atmospheric oxygen. Moreover, oxidative stress lasting for 5 h increased the binding, entry and survival of both C. jejuni isolates inside the macrophages. These results suggest that bacterial mechanisms involved in inactivating toxic oxygen products may enhance bacterial persistence within the phagocytes. Our results showed that campylobacter is efficiently internalized, but cannot remain viable for extended periods within murine macrophages. Bacteria were rapidly eliminated from macrophages within 24 h to 30 h post infection. Active killing of the bacteria by macrophages would explain why bacteraemia during Camyplobacter infection is not a common phenomenon. On the other hand, the differences noted between laboratories regarding the ability of C. jejuni to survive within phagocytes may be the consequence of the use of phagocytic cells of different origin and various bacterial isolates. In vitro studies of environmental regulation of bacterial virulence provide a number of important observations regarding the strategies used by pathogens to respond and survive under changing environmental conditions in vivo. However, additional work is required to determine the mechanism used by C. jejuni to alter its virulence factors and the precise role of macrophages in the development of campylobacteriosis. Acknowledgements The authors would like to thank the Ministry of Education, Science and Sport of the Republic of Slovenia and the Ministry of Science, Education and Sports of the Republic of Croatia for financing the bilateral Croatian–Slovenian project “Resistance and virulence of Campylobacter species”. Partial support was also provided through a research project (062-0621273-1235) financed by the Croatian Ministry and a PhD grant to A.K. from the Slovenian Ministry.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Survival of stress exposed Campylobacter jejuni in the murine macrophage J774 cell line Maja Šikić Pogačar a, Roberta Rubeša Mihaljević b, Anja Klančnik a, Gordana Brumini c, Maja Abram b, Sonja Smole Možina a,⁎ a b c
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia Department of Microbiology, Medical Faculty, University of Rijeka, Brace Branchetta 20, HR-51000 Rijeka, Croatia Department of Medical Informatics, Medical Faculty, University of Rijeka, Brace Branchetta 20, HR-51000 Rijeka, Croatia
a r t i c l e
i n f o
Article history: Received 29 February 2008 Received in revised form 27 October 2008 Accepted 9 November 2008 Keywords: Campylobacter jeuni Environmental stress Macrophage Survival
a b s t r a c t Although campylobacters are relatively fragile and sensitive to environmental stresses, Campylobacter jejuni has evolved mechanisms for survival in diverse environments, both inside and outside the host. Their survival properties and pathogenic potential were assessed after subjecting food and clinical C. jejuni isolates to different stress conditions. After exposure to starvation (5 h and 15 h of nutrient depletion), a temperature shock (3 min at 55 °C) or oxidative stress (5 h and 15 h of atmospheric oxygen) we studied the culturability, viability and capability of adhesion, internalization and survival within the in vitro cell culture model using J774 murine macrophages. Starvation severely impaired C. jejuni culturability, particularly after 15 h of nutrient depletion. The number of viable cells decreased by 30–40%. Starved bacterial cells also showed a lower capability of adhesion, internalization and survival within macrophages. Despite the reduced culturability and viability of the heat treated cells, C. jejuni efficiently adhered to, and entered murine macrophages. However, the number of heat treated cells started to decrease more quickly than non-stressed cells. Within 24 h post infection all the cells were killed. The bacterial mechanisms involved in inactivating toxic oxygen products may enhance bacterial persistence through increased binding, entry and survival of both oxidatively stressed C. jejuni isolates inside the macrophages. Oxygen exposure increased the internalization and intracellular survival, although the cells cannot remain viable for extended periods within murine macrophages. However, any prolongation of survival in macrophages may increase the probability of transmission of bacteria in the host organism and have further implications in the pathogenesis of campylobacteriosis. This indicates that environmental stress conditions may be involved. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Campylobacter jejuni is a gram negative, slender, curved, and highly motile rod-like bacterium. It has microaerophilic and thermophilic character with a requirement for reduced levels of oxygen and an optimal growth temperature of 42 °C, probably reflecting an adaptation to the avian gastrointestinal system. Healthy chickens carry these bacteria in their gut causing contamination of raw poultry meat, which is considered the major source of human food-borne campylobacteriosis (Park, 2002). C. jejuni and related campylobacters are relatively fragile and sensitive to environmental stresses like drying, heating, disinfectants, acidity and oxygen exposure. But, despite fastidious growth requirements and increased environmental sensitivity, C. jejuni is recognized as a leading cause of human gastroenteritis worldwide (Adak et al., 2005), indicating that it has developed some mechanisms for survival
⁎ Corresponding author. Tel.: +386 1 423 11 61; fax: +386 1 256 62 96. E-mail address: [email protected] (S. Smole Možina). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.11.010
in diverse environments, both inside and outside the host (Murphy et al., 2006; Young et al., 2007). Stress-responsive alternative sigma factors, including RpoS in gram-negative bacteria (Ibanez-Ruiz et al., 2000) and σB in gram-positive bacteria with a low proportion of guanine-cytosine content, contribute to regulation of large sets of genes (Petersohn et al., 2001). As reported for L. monocytogenes, exposure to sublethal stresses might enhance the organism's virulence, e.g., by activating the expression of σB-regulated virulence genes (Ferrierra et al., 2001; Kazmierczak et al., 2003). Exposure of L. monocytogenes to different environments may have additional effects on virulence-related characteristics (Garner et al., 2006). Campylobacters lack many of the adaptive responses to environmental stresses known to enable survival of other food-borne pathogens, including alternative sigma factor σB, that contributes to transcription of stress response and virulence genes (Park, 2002). In spite the high prevalence of C. jejuni in human bacterial gastroenteritis, the pathogenesis of infection is still poorly understood. Once inside the human digestive system, campylobacter colonizes and attacks the mucosa of the small and large intestine.
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Attachment to intestinal epithelial cells is an early and important step in the infectious process leading to bacterial internalization. Examination of intestinal biopsies of humans (Chang and Miller, 2006), in vivo studies in animal models (McOrist et al., 1989; Russell et al., 1993; Vučković et al., 1998), together with in vitro experiments using cultured intestinal epithelial cells (Friis et al., 2005; Kalischuk et al., 2007; Mihaljević et al., 2007) have clearly demonstrated that C. jejuni can invade non-phagocytic intestinal epithelial cells. Internalization of C. jejuni is reduced at low temperature (Konkel et al., 1992), as well as upon exposure to heat shock or starvation, but seems to be increased when pre-cultivated under aerobic conditions for 5 h (Mihaljević et al., 2007). Once within epithelial cells, C. jejuni can survive (Konkel et al., 1992; Watson and Galán, 2008) and multiply intracellularly (Mihaljević et al., 2007). Thereafter, C. jejuni can gain access to underlying tissues and reach different cellular receptors and professional phagocytic cells. It is known that survival of bacteria within phagocytic cells represents a strategy to evade the host defences and allows proliferation and dissemination of bacteria throughout the host. On the other hand, once inside the macrophage, bacteria must be able to overcome a variety of unfavourable conditions triggered by the host's killing mechanisms, such as oxidative products, nutrient limitation, acidic pH and other stimuli. There are contradictory reports regarding the ability of C. jejuni to survive within macrophages, depending on the macrophage cell type and C. jejuni strain. There is evidence that viable campylobacters can be recovered from human monocyte cultures after extended periods (Hickey et al., 2005; Kiehlbauch et al., 1985) and that catalase-producing strains have an enhanced chance of intramacrophage survival (Day et al., 2000). On the contrary, other data indicate that intraphagocytic survival is not a
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common phenomenon during campylobacter infection because human derived monocytes/macrophages kill phagocytosed bacteria within 24 to 48 h (Wassenaar et al., 1997). It has been found that C. jejuni is targeted on lysosomes and therefore cannot survive inside mouse primary bone marrow-derived macrophages (Watson and Galán, 2008). The aim of our study was to analyse whether submission to an environmental stress may modulate the virulence characteristics of Campylobacter and give it an advantage in surviving in macrophages. We initially examined the changes of culturability and viability of C. jejuni in response to temperature shock, starvation, and atmospheric oxygen pre-treatment. After stress exposure, in an in vitro cell culture model using J774 murine macrophages, changes in adhesion, internalization and intracellular survival of two C. jejuni isolates, one clinical and one from food, were studied. 2. Materials and methods 2.1. Bacterial strains, culture media and growth conditions A food (poultry meat) isolate (K49/4) and a clinical isolate obtained from a patient with severe diarrhea were identified by phenotypic and molecular techniques as described previously (Zorman and Smole Možina, 2002). The strains were stored at −80 °C in a brain heart infusion broth supplemented with blood and glycerol, and subcultured prior to subjecting them to experimental procedures. Microaerobical (O2 5%, CO2 10%, and N2 85%) growth in Preston broth at 42 °C for 9 h induced the entry of the culture into the exponential growth phase, as determined previously (Klančnik et al., 2006).
Fig. 1. Culturability and viability of C. jejuni clinical (A) and food (B) isolates of non-stressed and stress exposed C. jejuni. The results are presented as relative rate ± SD (*P ≤ 0.05).
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2.2. Stress conditions Starvation, high temperature and oxidative stress were selected as bacterial stress conditions. For starvation, campylobacters were harvested by centrifugation (12,000 ×g at 4 °C for 5 min), washed, resuspended in Ringer solution (KH2PO4, 5 mM (Kemika), which was used as a multiple nutrient starvation medium, and incubated for 5 or 15 h microaerobically at 42 °C. To achieve heat shock the cells were shifted from 42 to 55 °C for 3 min and cooled on ice before analysis. For oxidative stress, the cells were exposed to atmospheric oxygen for 5 or 15 h. Non-stressed bacterial culture, taken at the same time as the stressed culture, served as the control. For culturability, viability, adhesion and invasion the relationship between stressed and nonstressed control cultures (relative rate) represented the influence of individual stresses. 2.3. Assessment of bacterial culturability and viability Culturability (CFU mL− 1) was assessed on Karmali agar plates (Oxoid, UK) after serial sample dilutions and microaerobic incubation at 42 °C for 24 or 48 h. Culturability experiments were independently repeated three or more times and the results expressed as log10 CFU/mL relatively to the non-treated control at the same time.
Total and viable counts were assessed using the LIVE/DEAD® BacLight™ viability kit (L-7012, Molecular Probes, Eugene, Oregon, USA) and microscopically examined (Eclipse TE300, Nikon, super high pressure mercury lamp power supply, X-60 oil immersion fluorescent objective, Nikon digital camera DXM 1200, Nikon Programing Equipment Laboratory Imaging Ltd., LUCIA 4.60, Tokyo, Japan) using blue excitation light as described previously (Klančnik et al., 2006). Viability is given as the percentage of viable cells relative to the total abundance obtained in 20 microscopic fields per filter. Viability experiments were independently repeated at least twice. The results are expressed as viable cells relatively to the non-treated control at the same time. 2.4. Adhesion, invasion and intracellular survival in J774 macrophages J774 macrophages were grown in RPMI 1640 medium (Institute of Immunology, Zagreb, Croatia) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 000 UI/mL penicillin and 10 000 μg/mL streptomycin (Gibco, London, UK). The cells were grown in tissue culture Petri dishes at 37 °C in a 5% CO2 humidified atmosphere. For all assays, 24-well tissue culture trays were seeded with approximately 5.0 × 105 macrophages per mL, and incubated for 24 h. The bacterial number was determined spectrophotometrically (absorbance mode at
Fig. 2. Adhesion and invasion ability of C. jejuni clinical (A) and food (B) isolates to J774 macrophages after stresses exposure. The results are presented as relative rate ± SD (*P ≤ 0.05).
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a wavelength of 600 nm). Infection was carried out by inoculating C. jejuni at approximate MOI of 100. Infected monolayers were incubated for 2 h to allow adhesion and invasion to occur. Then, the monolayers were washed three times with RPMI without an antibiotic to remove unbound bacteria. For the bacterial invasion assay, a medium containing 100 μg/mL of gentamicin was added for 1 h to kill extracellular bacteria. Following this period, monolayers were lysed with cold distilled water, and the released internalized bacteria were enumerated by plating serial dilutions on blood agar plates. For intracellular survival analysis, the invasion period was monitored 3, 5, 10, 15, 24 and 29 h post infection. The total number of bacteria associated with the macrophages (adherent + engulfed) was determined simultaneously by performing an invasion assay but without gentamicin treatment. The difference between the total and the number of intracellular bacteria was calculated and the number of adherent C. jejuni was obtained. Results for adhesion and invasion are expressed as relative rate ± SD of log10 CFU/mL of adherent or internalized bacteria, and for intracellular survival as the mean ± SD of log10 CFU/mL of internalized bacteria, for three independent measurements.
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2.5. Statistical analysis Data on bacterial counts from different experimental groups were compared using one-way ANOVA and Tukey-b tests for post-hoc analysis. All statistical values were considered significant at the P level of 0.05. Statistical analysis was performed using Statistica for Windows version 6.1 (Statsoft Inc., Tulsa, OK, USA). 3. Results 3.1. Culturability and viability of C. jejuni exposed to different stress conditions The culturability of clinical (A) and food (B) isolates of C. jejuni was monitored after exposure to temperature shock, atmospheric oxygen or nutrient deficiency (Fig. 1). After exposure to heat, a significant loss of culturability was observed for both isolates, although the food isolate showed slightly higher thermal resistance in comparison to the clinical strain. Starvation severely impaired bacterial culturability, particularly after 15 h of storage in a low nutrient medium. On the contrary, bacterial cultivation properties were unchanged following exposure to atmospheric oxygen. In parallel with the culturability analysis, viability changes of C. jejuni isolates were observed (Fig. 1). The level of viable cells decreased approximately 30–40% during heat shock and after starvation for 15 h. A decline of 20% was noticed in cell starved for 5 h; all oxidative stresses were milder. 3.2. Influence of environmental stresses on Campylobacter adhesion, invasion and intracellular survival
Fig. 3. Intracellular survival of control and stresses exposed C. jejuni, clinical (A) and food (B) isolates in J774 macrophage cell line. The bacterial number was determined by the CFU assay. Each point represents the log10 of the mean ± SD CFU/mL (*P ≤ 0.05).
Bacterial adhesion to, and uptake by J774 macrophages was assessed by plate-count assay after C. jejuni exposure to starvation, heat stress or atmospheric oxygen and compared to a non-stressed control. As shown in Fig. 2, there was no significant difference in the number of adhering or engulfed campylobacters after temperature shock in the case of both the clinical (A) and food (B), isolates. On the contrary, storage in a low nutrient medium, particularly for 15 h, significantly impaired bacterial adhesion as well as invasion properties. However, the food isolate was found to be more susceptible to starvation because a significant reduction in the number of adhering and ingested bacteria occurred already after 5 h of incubation in a low nutrient medium. Prolonged starvation for 15 h significantly reduced bacterial internalization. While 5 h exposure to atmospheric oxygen enhanced bacterial virulence properties according to the increased number of bound and intracellular bacteria, prolonged aeration (for 15 h) had no impact on the clinical strain but significantly decreased the adherence and invasion capability of the food isolate. To determine the role of selected stress conditions on C. jejuni survival within phagocytes in vitro, cultured murine J774 macrophages were infected with the clinical (Fig. 3A) and food (Fig. 3B) isolates and survival kinetics were analysed over a 48 h period. Both strains exhibited similar survival characteristics, undergoing a progressive decrease in the number of intracellular bacteria. It should be noted that 24 or 29 h post infection, no intracellular bacteria were recovered, indicating extensive killing by the macrophages. Pre-exposure of campylobacters to environmental stresses did not significantly influence the length of intramacrophage survival. However, considerable differences in the number of intracellular bacteria were observed. Bacterial starvation significantly decreased the survival period and the number of both C. jejuni isolates in J774 macrophages and correlated with the duration of pre-cultivation in the nutrient-poor medium. When the starvation period lasted for 15 h, C. jejuni was capable of surviving inside macrophages for only 10 h in the case of the food isolate, and 15 h in the case of the clinical isolate. Heat shock did not affect the initial phase of intracellular growth of C. jejuni, when the
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bacterial load was similar to the control level. However, the number of bacteria within macrophages started to decrease as early as 5 h post infection. Interestingly, our results confirmed that despite the microaerophilic nature of Campylobacter, pre-exposure to atmospheric oxygen enhanced its intramacrophage survival. When compared to the non-stressed control, viable clinical and food strains were recovered in higher numbers over the entire period of infection. On the other hand, prolonged oxidative stress (for 15 h), increased the sensitivity of the food strain to macrophage killing, since a lower CFU was detected, while the intracellular growth of the clinical isolate was not significantly affected. 4. Discussion Through the course of food borne infection food pathogens are subjected to host-imposed stress conditions, including the acidic stress of the stomach and then the oxidative stress of the host cell vacuole. Many intracellular food-borne bacteria capable of surviving in the intracellular compartment like Yersinia enterocolitica (Yamamoto et al., 1994) and Salmonella typhimurium (Ibanez-Ruiz et al., 2000) produce large amounts of stress proteins after phagocytosis. Their survival inside various cells, including phagocytes, has been proposed as a key element in the pathogenesis and manner of persistence in certain infections. Additionally, the mechanism used for defence against a single stress may provide bacteria cross-protection towards other unrelated hostile conditions. Bacterial cells can adapt to normally unfavourable growth conditions following a brief exposure to mild stress (Hill et al., 1995; Klančnik et al., 2008). It has been confirmed for Campylobacter spp. that submission to an initial stress may improve bacterial resistance to a second successive stress, enhancing their pathogenic characteristics (Murphy et al., 2003). Consequently, alterations in the growth environment of a bacterium may alter the expression of a variety of virulence factors (Biswas et al., 2007). The intracellular environment of a macrophage is one of the most hostile surroundings encountered by an invading bacterium. Therefore, the objective of this study was to examine in vitro in the cellculture model the efficiency of C. jejuni uptake into macrophages and to determine how long the bacteria can survive inside these cells. In addition, we were interested whether starvation, heat shock and atmospheric oxygen exposure induce changes in culturability/viability, as well as internalization and intramacrophage survival of clinical and food C. jejuni isolates. Starvation is the most significant factor in C. jejuni survival, coccoid cell formation and especially culturability (Klančnik et al., 2008). Also according to our results, starvation was the most powerful stress condition, which significantly affected C. jejuni culturability and viability, particularly after 15 h of nutrient depletion. This is consistent with the evidence that, unlike most other bacteria, C. jejuni does not exhibit increased survival during long periods of food deprivation, such as upon entry into the stationary phase (Kelly et al., 2001; Klančnik et al., 2006). When the effect of nutrient limitation on bacterial virulence was examined, starved bacterial cells of both strains showed a lower ability of adhesion, internalization and survival within macrophages. Previous studies confirmed that lack of nutrients and aging in Campylobacter induce disruption of both the inner and outer membrane structures and their functions (Kelly et al., 2001). The appearance of such sublethally injured cells, which prevail in the bacterial population, may be responsible for the significant loss of virulence and inhibition of bacterial growth. In its contamination cycle C. jejuni encounters a wide range of temperatures; during food processing, while entering the host and in the environment. Once exposed to 55 °C, a significant decrease of growth on agar, as well as in viability was detected. It is well known that immediately following heat shock C. jejuni increases the expression of a number of proteins (Parkhill et al., 2000). Many of
these proteins have been confirmed to play a role in bacterial–host interactions (Konkel et al., 1998) and could promote bacterial adherence and uptake in macrophages. However, contrary to expectation, C. jejuni efficiently adhered to, and entered murine macrophages, indicating that 3 min of heat shock was probably not enough for increased protein synthesis and that further incubation to monitor protein level changes, as suggested by Konkel et al. (1998), should be performed. The growth of heat treated bacteria appeared unaffected by the host intracellular milieu within the first 5 h post infection. However, afterwards both strains declined significantly in number and most of the intracellular bacteria were rapidly killed. Thus, the heat shock difference observed in Fig. 2 may simply be due to surface structural changes rather than increased protein production. Colonization of the host is a process involving adaptation of C. jejuni to different atmospheric oxygen concentrations. Although Campylobacters are generally considered microaerophilic, many reports indicate that C. jejuni has the ability of long-term aerobic adaptation (Murphy et al., 2003; Jones et al., 1993). Our results confirmed a similar outcome during exposure of campylobacter to oxidative stress. Microscopic counts of viable bacteria, as well as CFU numbers, exhibited no significant change after storage in atmospheric oxygen. Moreover, oxidative stress lasting for 5 h increased the binding, entry and survival of both C. jejuni isolates inside the macrophages. These results suggest that bacterial mechanisms involved in inactivating toxic oxygen products may enhance bacterial persistence within the phagocytes. Our results showed that campylobacter is efficiently internalized, but cannot remain viable for extended periods within murine macrophages. Bacteria were rapidly eliminated from macrophages within 24 h to 30 h post infection. Active killing of the bacteria by macrophages would explain why bacteraemia during Camyplobacter infection is not a common phenomenon. On the other hand, the differences noted between laboratories regarding the ability of C. jejuni to survive within phagocytes may be the consequence of the use of phagocytic cells of different origin and various bacterial isolates. In vitro studies of environmental regulation of bacterial virulence provide a number of important observations regarding the strategies used by pathogens to respond and survive under changing environmental conditions in vivo. However, additional work is required to determine the mechanism used by C. jejuni to alter its virulence factors and the precise role of macrophages in the development of campylobacteriosis. Acknowledgements The authors would like to thank the Ministry of Education, Science and Sport of the Republic of Slovenia and the Ministry of Science, Education and Sports of the Republic of Croatia for financing the bilateral Croatian–Slovenian project “Resistance and virulence of Campylobacter species”. Partial support was also provided through a research project (062-0621273-1235) financed by the Croatian Ministry and a PhD grant to A.K. from the Slovenian Ministry.
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