Aim: To study stress resistance mechanisms in Campylobacter spp.
Methods and Results: Campylobacter strains were grown to the appropriate phase in Brucella broth. The cells were diluted into either cell-free spent medium (obtained by filtration of a grown culture) or a freshly prepared medium and the pH reduced to 4·5, a lethal pH value. At suitable time intervals survivors were enumerated on Campylobacter blood free selective agar base. The cell-free spent medium from mid-exponential and stationary phase had a protective effect on acid and thermal stress in Campylobacter jejuni CI 120, a natural isolate. The protective effect of the extracellular compound was not significantly inactivated by boiling, but was inactivated by proteinase.
Conclusions: The present study suggests that a protein (or proteins) accumulated by C. jejuni CI 120 during growth may play an active role in the induction of stress responses and that this protein is heat stable.
Significance and Impact of the Study: The results indicate that C. jejuni CI 120, a natural isolate, has the ability to use extracellular signalling mechanisms to induce tolerance to stress factors. This is a major advancement in the understanding of the physiological basis for survival of C. jejuni in the environment.
Campylobacter spp. are the most common bacterial cause of food poisoning world-wide. While there are 15 different species, of which 12 are pathogenic to humans (Lastovica and Skirrow 2000), 95% of bacterial gastroenteritis in humans is caused by C. jejuni and C. coli (Park 2002). Being very fastidious organisms, they are very sensitive to environmental stress and require a strictly microaerobic growth atmosphere of 5% O2, 10% CO2 and 85% N2. They appear to lack most of the stress response factors necessary for survival of other organisms in an adverse environment such as the global stationary phase stress response factor RpoS, the oxidative stress response factor SoxRS and other stress response factors such as Lrp, ProU, CspA and RpoH (Park 2002). Considering the prevalence of illness caused by Campylobacter spp. and the environmental sensitivity of the organism, the indications are that they have developed mechanisms for survival of adverse conditions that are not commonly found in other organisms. Although the entry into a viable nonculturable state (Rollins and Colwell 1986), the transition from rod to coccoid shape (Moran and Upton 1987) and a high degree of genetic heterogeneity among strains (Wassenaar et al. 2000) have been suggested as possible survival mechanisms, there is a general lack of understanding of the physiology of campylobacters. There is a suggestion that they lack the adaptive responses found in other organisms (Park 2002), which further emphasizes the importance of survival mechanisms not yet identified.
Rowbury (2001) has described the detailed physiological characteristics of a two-component extracellular regulatory system in Escherichia coli where an extracellular compound produced by growing cells can be converted to an induction compound by stress factors, leading to a tolerance response. Nikolaev (1996) has also described extracellular protectants in E. coli, while Vorobjeva et al. (1999) have isolated an extracellular nucleoprotein from Propionibacterium freudenreichii subsp. shermanii that has a protective effect on stressed E. coli.
The aim of the present study was to investigate novel mechanisms of survival in Campylobacter spp.
Materials and methods
Organisms and growth conditions
C. jejuni CI 120, CI 195, CN 107, NCTC 11351 and NCTC 81116 were used during these studies. C. jejuni CI 120 was isolated from poultry during processing and CI 195 was isolated at the end of processing. Both were identified by API Campy (BioMérieux, Marcy l'Etoile, France) and by a PCR/DNA probe membrane-based colorimetric assay (O'Sullivan et al. 2000). C. jejuni CN 107 (campynet collection strain) was obtained from Bob Madden (Queen's University, Belfast). Strains 11351 and 81116 were obtained from the National Collection of Typed Cultures (Central Public Health Laboratory, London, UK). All strains were maintained at −20°C in Brucella broth (Difco) supplemented with 15% (v/v) glycerol. C. jejuni strains were routinely grown at 42°C in a multi-gas incubator under microaerobic conditions (5% O2, 10% CO2 and 85% N2). Cultures were routinely subcultured onto Campylobacter blood free agar base (Oxoid) from frozen stocks prior to being subjected to test conditions.
Cell-free spent medium
To obtain a cell-free spent medium the cells were grown to the appropriate phase in filter sterilized Brucella broth. The culture was then aseptically filtered through a low protein binding 0·2 μm minisart plus filter (Sartorius, Göetingen, Germany) and the supernatant collected aseptically.
Assay of acid tolerance
Inactivation curves were performed in cell-free spent medium and freshly prepared medium. Cells were grown to mid-exponential or stationary phase and resuspended (1 : 10 dilution) in the different media. The pH was adjusted to 4·5 by direct addition of 0·5 m HCl and the cultures incubated at 42°C. Viable cell counts were performed immediately prior to the pH adjustment, immediately after the pH adjustment and at suitable time intervals thereafter. Serial dilutions were performed in maximum recovery diluent (Oxoid), and plate counts were performed by spreading diluted or undiluted cell suspensions on Campylobacter blood free selective agar base, which were incubated for 48 h at 42°C under microaerobic conditions.
Assay of heat tolerance
C. jejuni CI 120 was grown to the desired growth phase in Brucella broth at 42°C under microaerophilic conditions. Cell-free spent medium or freshly prepared medium (9 ml) was aseptically transferred to 15 ml sterile glass vials sealed with crimp caps containing a silicone septum (Carl Stuart Limited, Whitestown, Ireland). The vials were immersed in an oil-bath at 55°C (±0·1°C) for 10 min, to allow equilibration of temperatures. Using a sterile syringe needle, 1 ml of the bacterial culture was added through the silicone septum. The suspension was mixed for 5 s and 0·5 ml of sample removed at suitable time intervals and immediately cooled in iced water for 1 min before viable cell counts performed as described above.
Characterization of extracellular component
In order to perform preliminary characterization of the extracellular component, the cell-free spent medium was heat treated and exposed to proteinase. For heat treatment, cell-free spent medium was incubated in a boiling waterbath for 10 min. For proteinase treatments, the cell-free spent medium was incubated with agitation at 37°C with 2 U ml−1 of proteinase K-agarose (Sigma) for 1 h. The proteinase was subsequently removed by filtration. Fresh medium was used as a control in both the above experiments.
Reproducibility of results
All experiments were undertaken at least in triplicate and the results expressed are the average with the standard error of the mean shown as error bars.
Assay of acid tolerance
Figure 1 shows that throughout the time course of the experiment, when acid stress (pH 4·5) was applied in cell-free spent medium, mid-exponential and stationary phase cells of C. jejuni CI 120 had as much as 1000-fold better survival than when the stress was applied in freshly prepared medium. However, when mid-exponential or stationary phase cells were resuspended in cell-free spent medium from a different phase there was no protective effect. Neither was there any protective effect from lag phase spent medium (data not shown). All further experiments undertaken were with cell-free spent medium that was from the same phase as the cells being examined.
Assay of heat tolerance
C. jejuni CI 120 was exposed to a heat challenge of 55°C in cell-free spent medium or fresh medium. Figure 2 shows that spent medium can increase tolerance to heat stress. Thermal inactivation at 55°C resulted in almost a 100-fold greater survival of mid-exponential and stationary phase cultures resuspended in cell-free spent medium when compared with that resuspended in fresh medium.
Characterization of the extracellular component
The cell-free spent medium was diluted with fresh medium to 25, 50 or 75%. As the dilution of the cell-free spent medium increased the protective effect decreased.
The cell-free spent medium was heat treated by boiling for 10 min and the cells resuspended in this medium and challenged at pH 4·5 (Table 1). The degree of protection of the cell-free spent medium was only partially reduced by this heat treatment, suggesting that the extracellular component is heat stable.
Table 1. The percentage survival at pH 4·5 of Campylobacter jejuni CI 120 resuspended (1 : 10 dilution) in fresh medium, cell-free spent medium and boiled cell-free spent medium. All experiments were undertaken in triplicate
Percentage survival of Campylobacter jejuni in various media
89 ± 7·77
89 ± 6·87
99 ± 3·87
6 ± 1·16
54 ± 2·85
41 ± 1·78
0·04 ± 0·01
22 ± 6·79
5 ± 1·94
0 ± 0
3 ± 0·08
0·2 ± 0·08
Stationary phase cells
98 ± 5·33
96 ± 13·64
121 ± 31·87
8 ± 4·73
63 ± 2·18
15 ± 1·80
2 ± 2·22
14 ± 2·23
5 ± 2·28
0·04 ± 0·01
1·2 ± 0·21
0·2 ± 0 ·05
Cells resuspended in proteinase-treated cell-free spent showed similar death kinetics to cells resuspended in fresh medium (Fig. 3), indicating that the compound responsible for the protective effect is a protein.
Protective effect of cell-free spent medium from C. jejuni CI 120 on other strains
Survival of two strains of C. jejuni, NCTC 11351 and NCTC 81116, was studied in cell-free spent medium obtained from cells of strain CI 120 grown to mid-exponential phase. Similar to strain CI 120 itself, both strains showed 50–100-fold increased acid tolerance in the cell-free spent medium, compared with fresh medium (data not shown).
Protective effect of cell-free spent medium with other strains of C. jejuni
Cell-free spent medium was obtained from the growth to mid-exponential phase of four strains of C. jejuni, two culture collection strains (NCTC 11351 and NCTC 81116) and two natural isolates (CN 107 and CI 195). The results showed that the two culture collection strains did not produce any extracellular protective effect as survival was similar in fresh and cell-free spent medium (data not shown). The two natural isolates did produce an extracellular protective compound. Survival of these strains in cell-free spent medium was increased by about 50-fold, when compared with survival in fresh medium (Fig. 4).
Campylobacter spp. are the most common cause of bacterial food poisoning, but yet they are the most fastidious and stress sensitive of the common food-borne pathogens (Park 2002). However, their mechanisms of survival must be such that they can survive on food, in the environment and be able to survive the low pH of the stomach. We have determined the presence of an extracellular component secreted during growth that induces acid and heat stress tolerance in C. jejuni (Figs 1 and 2). This response was not the induction of the typical stationary phase RpoS global response mechanism as Campylobacter spp. does not contain the genes for the RpoS mechanism. In addition to this, contrary to when RpoS is activated, stationary phase cells of C. jejuni are considerably more acid sensitive than mid-exponential phase cells (Kelly et al. 2001).
The extracellular component appears to be phase specific. When cells from either mid-exponential or stationary phase were resuspended in cell-free spent medium of a different phase there was no protective effect. This result suggests that stationary phase cells produce a slightly modified or different extracellular component than mid-exponential phase cells or that the cell receptors are different in each stage of growth. There was also no protective effect observed in cell-free spent medium obtained from lag phase cells. It is possible that the active component was not produced during this early stage of growth.
Processes such as heating and acidity are commonly used to control food-borne pathogens. The identification of an extracellular compound in Campylobacter spp. that can induce a tolerance response to both of these stresses could be a significant step towards understanding the physiology of survival in Campylobacter spp. and should help towards the development of control strategies.
The active component appears to be heat stable as heat treatment for 10 min only reduced the protective effect provided against acid challenge (Table 1). Cells resuspended in proteinase-treated cell-free spent medium showed death kinetics similar to that of cells resuspended in fresh medium (Fig. 3). This suggests that the loss of the protective effect could be due to inactivation of extracellular protein(s) in the cell-free spent medium.
It is interesting that the cell-free spent medium from strain CI 120 conferred acid tolerance to the other strains of C. jejuni tested. This may have important implications in the environment. As not all strains produce the protective component, production by one strain could give protection to all strains present. It is also interesting that of the strains tested only the natural isolates produced the protective effect. This raises the question as to whether the culture collection strains have lost the ability to produce the compound, or whether the natural isolates have survived in the environment as a result of their ability to produce an extracellular protective compound.
Intracellular processes, such as transcription and translation, are more commonly proposed as inducible mechanisms of survival in bacterial cells (Neidhardt et al. 1990). In contrast, quorum sensing is an extracellular process of cell-to-cell signalling that contributes to enhanced survival of environmental stress. Quorum sensing was first characterized in the marine bacteria Vibrio harveyi and V. fischeri (Nealson et al. 1970; Nealson and Hastings 1979). Campylobacter spp. has been shown to contain the LuxS homologue, which has been suggested to play a role in the regulation of motility in Campylobacter (Elvers and Park 2002). They have also suggested that this system could be an important regulatory mechanism for basic physiological functions and possibly virulence factors. It is unlikely that quorum sensing is involved in the increased tolerance observed. Cell numbers in the fresh and spent medium were similar and the cell-free spent medium was prepared from stress free growth conditions. There was no induction period with mild stress during growth, therefore, the tolerance effect observed is also totally independent of any adaptive tolerance response.
Other extracellular proteins that confer a protective effect have been reported for E. coli and P. freudenreichii (Nikolaev 1996; Vorobjeva et al. 1999). Whether the mechanism of action of the C. jejuni CI 120 extracellular component is similar to either of these remains to be elucidated. However, as the protective effect is considerably greater than either of these reported effects, it is likely to have a different mode of action.
In conclusion, an extracellular protein that provides protection to acid and heat stress has been identified in C. jejuni. This novel mechanism of stress tolerance contributes to our understanding of the survival of C. jejuni under environmental conditions.
This work was partly financed by the Dairy Industry in Ireland. C. Murphy is the recipient of a Teagasc Walsh Fellowship. We thank Bob Madden (Department of Food Science, Queen's University, Belfast) for the C. jejuni strain CN 107.