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Keywords:

  • Campylobacter;
  • chicken;
  • high hydrostatic pressure;
  • milk

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To compare pressure resistance between strains of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus, and to investigate the effect of suspending medium on pressure resistance of sensitive and more resistant strains.

Methods and Results:  Six strains of C. jejuni and four each of C. coli, C. lari and C. fetus were pressure treated for 10 min at 200 and 300 MPa. Individual strains varied widely in pressure resistance but there were no significant differences between the species C. jejuni, C. coli and C. lari. Campylobacter fetus was significantly more pressure sensitive than the other three species. The pressure resistance of C. jejuni cultures reached a maximum at 16–18 h on entry into stationary phase then declined to a minimum at 75 h before increasing once more. Milk was more baroprotective than water, broth or chicken slurry but did not prevent inactivation even of a resistant strain at 400 MPa.

Conclusions:  Pressure resistance varies considerably between species of Campylobacter and among strains within a species, and survival after a pressure challenge will be markedly influenced by culture age and food matrix.

Significance and Impact of the Study:  Despite the strain variation in pressure resistance and protective effects of food, Campylobacter sp. do not present a particular problem for pressure processing.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

High hydrostatic pressure has proved to be one of the more successful of the new nonthermal food preservation technologies and several products using this technology are now available commercially (San Martín et al. 2002). Initial food applications were mainly for fruit juices and other acid products but there is growing commercial interest in applying pressure technology to meat products and ready-to-eat foods. In order to ensure the safety of pressure-processed foods, it is essential to understand the effects of pressure on the behaviour of food-borne pathogens, and a considerable body of relevant data has accumulated in recent years (Farkas and Hoover 2000). However, although Campylobacter spp. are the commonest bacterial cause of food-borne gastroenteritis in developed countries, we currently know very little about the response of these organisms to high pressure. Many members of the genus Campylobacter are capable of causing gastrointestinal disease in humans but the most important species are Campylobacter jejuni and Campylobacter coli and to a lesser extent Campylobacter lari which together are responsible for more than 95% of cases. Campylobacter jejuni and C. coli have minimum growth temperatures of around 30°C and are, therefore, unlikely to be able to multiply in food under most circumstances (Stern and Line 2000). Nevertheless, because the infectious dose is low, survival after pressure treatment, even in low numbers, would pose a significant risk to health. With the current interest in extending the range of foods suitable for pressure processing, it would seem particularly important to understand the factors affecting pressure resistance of these common food-borne pathogens.

Shigehisa et al. (1991) studied the inactivation of a single strain of C. jejuni in pork slurries by high pressure and concluded that these organisms were more pressure sensitive than Gram-positive cocci but of similar resistance to Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium. More recently, Solomon and Hoover (2004) examined the pressure resistance of C. jejuni strains ATCC 35919 and ATCC 35921 in broth and food substrates and concluded that treatment at 400 MPa for 10 min was sufficient to inactivate C. jejuni in both model broth systems and in foods. The two strains behaved very similarly in broth but strain ATCC 35919 seemed more resistant in food systems. Apart from these studies there is little information on the variation in pressure resistance between species of Campylobacter, or among strains of the same species. The aim of this work was to compare pressure resistance between strains of C. jejuni, C. coli, C. lari and Campylobacter fetus and to investigate the effects of growth phase and suspending medium on pressure resistance.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Organisms and growth conditions

Campylobacter jejuni NCTC 11351, C. jejuni NCTC 11349, C. jejuni NCTC 11168, C. jejuni NCTC11392, C. jejuni NCTC 11322, C. jejuni NCTC 10983, C. coli 11350, C. coli NCTC 11366, C. lari NCTC 11352, C. lari NCTC 11457, C. lari NCTC 11937, C. lari NCTC 11844, C. fetus ssp. fetus NCTC 10842, C. fetus ssp. fetus NCTC 5850, C. fetus ssp. fetus NCTC 10348 and C. fetus NCTC ssp. venerealis 10354 were obtained from the National Collection of Type Cultures, Colindale, UK. Campylobacter coli UA585, originally isolated from a diarrhoeic pig, was kindly provided by D.E. Taylor, University of Alberta, Edmonton, Canada; C. coli BM2509 was from the departmental culture collection. All strains were stored at −70°C in Microbank vials (ProLab Diagnostics, Neston, UK).

The liquid growth medium for Campylobacter sp. consisted of Brucella broth (Difco, East Moseley, UK) containing one vial of Campylobacter growth supplement (SR84 Oxoid, Basingstoke, UK) per 500 ml (BBFBP). The supplement contains sodium pyruvate, sodium metabisulphite and ferrous sulphate, each at 0·125 g l−1 to reduce oxidative stress. The agar plating medium consisted of Mueller–Hinton agar containing one vial of FBP supplement per 500 ml (MHAFBP). Maximum recovery diluent (MRD) and phosphate-buffered saline were obtained from Oxoid. Campylobacter cultures were prepared as described previously (Martínez-Rodriguez et al. 2004). Briefly, cells were grown from frozen stock in BBFBP under microaerobic conditions in a Variable Atmosphere Incubator (VAIN; Don Whitely, Otley, UK) at 39°C and subcultured once before inoculating into the experimental culture. Cultures were incubated on a shaking platform at 150 rev min−1 for 6 h to produce exponential phase cells, and for between 16 and 96 h for stationary phase cells, as specified in the text. When comparing pressure resistance between strains cells were normally obtained from cultures between 16 and 18 h old, containing a viable population of between 5 × 108 and 1 × 109 CFU ml−1. This coincides with the point on the growth curve where resistance is expected to be at a maximum (Martínez-Rodriguez and Mackey 2005). In the case of C. fetus 10354 and C. fetus 10348 which grew more slowly than the other strains, cells were collected somewhat later, after 24 h incubation, in order to be in similar physiological state.

Pressure treatment

Volumes of culture (1·5 ml) were pressure-treated in a Foodlab Plunger Press apparatus (Stansted Fluid Power, Stansted, UK) as described by Pagán and Mackey (2000). All pressure treatments were carried out at ambient temperature (c. 20°C). The rise in temperature in the transmitting fluid, measured with a thermocouple, was approx. 3·3°C per 100 MPa so the temperature in the sample chamber will have risen to about 30°C at 300 MPa and 33°C at 400 MPa.

Preparation and inoculation of food substrates and water

One millilitre of a stationary phase culture was centrifuged at 10 000 g for 15 min and the pellet resuspended in 1 ml of deionized distilled water, or ultra-high temperature milk purchased from a supermarket. Chicken meat slurry was prepared as follows: a whole chicken was obtained from a local supermarket, the breast skin was flamed and removed with a sterile scalpel, and the underlying meat was removed and placed in a sterile blender; the meat was inoculated with broth culture to give approx. 108 CFU g−1 and then homogenized for 2 min at room temperature. One-millilitre portions of inoculated milk, water or chicken meat were pressure treated as described above.

Viable counts

Serial decimal dilutions were prepared in MRD and 20-μl volumes were spread onto duplicate fresh plates of MHAFBP. The number of colony-forming units was assessed after plates had been incubated at 39°C in the VAIN for a minimum of 48 h. Comparisons of pressure resistance between species and strains were made using the Analysis of Variance program of SPSS for Windows (version 11·5, SPSS Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The influence of growth stage on pressure resistance of C. jejuni NCTC 11351

To determine whether pressure resistance varied during growth of C. jejuni 11351, samples were taken during exponential growth and at different times during stationary phase and exposed to a pressure treatment of 250 MPa for 10 min. Figure 1 shows a typical growth curve during which cell numbers increase to a maximum in stationary phase then decline to a minimum at 72 h before increasing once more. The lower curve shows survival levels after a pressure challenge at 250 MPa. Resistance to pressure reached a maximum in early stationary phase (16 and 18 h) then declined to a minimum at 72 h. A second increase in resistance to pressure then occurred that coincided with re-growth in the culture. Survival values are expressed as surviving fractions hence the plotted values represent real changes in resistance and do not simply reflect differences in the initial cell concentration.

image

Figure 1. Changes in pressure resistance at different phases of growth. Growth curve of Campylobacter jejuni NCTC 11351 (•). Survival of cells removed at different phases of growth and pressure treated for 10 min at 250 MPa (bsl00084). Data from a single experiment

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Variation in pressure resistance between species and strains of Campylobacter

Differences in survival between strains and species of Campylobacter after treatment of stationary phase cells at 200 MPa are shown in Table 1. Analysis of variance revealed that there were no significant differences in pressure resistance between C. jejuni, C. coli and C. lari. Most strains of these species were relatively resistant to pressure at 200 MPa and many strains underwent a reduction in number of only 1 log10 unit or less. Campylobacter fetus strains were significantly more sensitive than the other species tested (P < 0·05) showing log reductions of 3·6 or more log units. Analysis of differences between strains (excluding C. fetus) showed that C. jejuni 11322 and C. coli BM2509 were significantly more sensitive than all other strains tested (P < 0·05).

Table 1.  Variation in survival of Campylobacter strains after treatment at 200 MPa for 10 min
StrainN*Log reduction in countMeanMean for species
  1. *Number of replicates.

  2. †Calculated minimum reduction where no survivors detected.

C. jejuni 1135120·0, 0·00·00·7
C. jejuni 1134920·2, 0·60·4
C. jejuni 1116820·3, 0·20·3
C. jejuni 1139220·4, 0·50·5
C. jejuni 1132222·9, 2·62·8
C. jejuni 1098320·2, 0·50·4
C. coli 1136621·0, 1·41·21·1
C. coli UA58520·8, 0·70·8
C. coli Bm250922·3, 2·52·4
C. coli 1135020·0, 0·30·2
C. lari 1135220·0, 0·00·00·5
C. lari 1145720·0, 0·10·1
C. lari 1193720·6, 0·40·5
C. lari 1184421·5, 1·41·5
C. fetus 1084223·5, 3·73·64·0
C. fetus 585024·1, 4·54·3
C. fetus 103542>6·1, >6·7†>6·4
C. fetus 103481>5·8>5·8

Survival levels after treatment at 300 MPa were much lower than at 200 MPa, as expected (Table 2), and only five strains yielded viable counts above the detection limit. This precluded statistical analysis, but it is obvious that certain strains were much more resistant than others, particularly C. jejuni 11351, C. lari 11352 and C. lari 11457.

Table 2.  Variation in survival of Campylobacter strains after treatment at 300 MPa for 10 min
StrainN*Log reduction in countMean
  1. *Number of replicates.

  2. †Calculated minimum reduction where no survivors detected.

C. jejuni 1135122·5, 2·42·5
C. jejuni 113492>7·0, >7·2>7·1†
C. jejuni 111682>7·0, > 7·2>7·1
C. jejuni 113922>7·1, >7·2>7·2
C. jejuni 113222>7·2, >7·0>7·1
C. jejuni 109832>7·1, >7·2>7·2
C. coli 113662>7·3, >7·1>7·2
C. coli UA58526·1, 5·65·9
C. coli Bm25092>6·9, >7·1>7·0
C. coli 1135025·5, 5·75·6
C. lari 1135223·0, 2·32·7
C. lari 1145721·0, 1·31·2
C. lari 119372>7·4, >7·4>7·4
C. lari 118442>7·3, >7.>7·3
C. fetus 108422>6·7, >6·9>6·8
C. fetus 58502>7·0, >7·1>7·1
C. fetus 103542>6·1, >6·7>6·4
C. fetus 1034815·85·8

Effect of different substrates on the survival of C. jejuni to HHP

Stationary phase cells of C. jejuni 11322 and C. jejuni 11351 were suspended in BBFBP, water, milk or chicken slurry, and subjected to a high-pressure treatment of 200 MPa for 10 min. These two strains represented pressure sensitive and pressure resistant types. Campylobacter jejuni 11351 showed little reduction in viability at this pressure in any of the substrates (Fig. 2). Surprisingly, the greater pressure resistance of this strain was retained even when suspended in water. The more sensitive C. jejuni 11322 survived less well in all substrates: when cells were resuspended in BBFBP, water or chicken, a decrease of more than 2 log10 units was observed whereas in milk a decrease of only about 1·5 log10 unit was seen. This small but consistent baroprotective effect of milk was investigated further.

image

Figure 2. Effect of different suspending media on pressure resistance of Campylobacter jejuni NCTC 11322 (□) and C. jejuni NCTC 11351 (bsl00001). Stationary phase cells were pressure treated at 200 MPa for 10 min in BBFBP, water, milk or chicken meat slurry as indicated. Data are mean values from two independent experiments in which the mean difference between replicates was 0·17 log units

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Protective effect of milk on the survival of C. jejuni after different high-pressure treatments

The survival of C. jejuni 11351 and C. jejuni 11322 in milk was compared with that in BBFBP after treatment at different pressures (Fig. 3). Campylobacter jejuni 11351 was almost completely resistant to treatments of 200 and 250 MPa in both BBFBP and milk, but at 300 MPa, a reduction of approx. 3 log10 units occurred in BBFBP, while a decrease of only about 0·4 log10 units was seen in milk. The baroprotective effect of milk was more obvious with the weaker C. jejuni 11322. In this case, mean log10 reductions of 2·0, 3·9 and 6·7 occurred following treatment of cells in BBFBP at 200, 250 and 300 MPa, respectively, whereas for cells suspended in milk, the corresponding reductions were only 0·6, 0·6 and 1·0 log10 units. For both strains, no survivors were detected when cells were treated at 400 MPa, irrespective of whether cells were suspended in BBFBP or milk.

image

Figure 3. Baroprotective effect of milk at different pressures. Cells of (a) Campylobacter jejuni NCTC 11351 or (b) C. jejuni 11322 were pressure treated for 10 min at 200, 250 and 300 MPa in milk (bsl00001) or BBFBP (□). Data are mean values from two independent experiments in which the mean difference between replicates was 0·42 log units

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Campylobacter spp. are generally regarded as being more sensitive to heat, cold, drying and acid stresses than other common food-borne bacterial pathogens (Stern and Line 2000) and this work confirms that this sensitivity also extends to pressure treatment. The magnitude of the decrease in viability at 200 MPa of stationary phase cells of Campylobacter sp. is similar to that seen in exponential phase cells of other Gram-negative bacteria such as Escherichia coli (Benito et al. 1999; Pagán and Mackey 2000; Casadei et al. 2002). This is perhaps not surprising as C. jejuni and probably other Campylobacter species lack the rpoS-encoded sigma factor, found in most other bacteria, that regulates the general increase in stress resistance that takes place as cells enter stationary phase (Parkhill et al. 2000; Kelly et al. 2001).

Despite this general sensitivity to pressure there were clear differences between species of Campylobacter and among strains of the same species. Campylobacter fetus was the most sensitive species examined and this is consistent with its generally fastidious nature. Strains of C. lari were slightly more pressure resistant on average but this was not statistically significant and more strains would have to be examined to decide whether this is a general property of C. lari.

The differences in pressure resistance between strains of a species were quite marked. For example differences in survival between the least and most pressure sensitive strains of C. jejuni and C. lari varied by approx. 104- and 106-fold, respectively, after treatment at 300 MPa for 10 min. The strains identified here may be useful in studying the physiological basis of the differences in stress resistance in Campylobacter sp. and it will be interesting to discover whether the pressure sensitive and resistant strains also differ in their resistance to other stresses.

When C. jejuni enters stationary phase it does not show the usual marked increase in resistance to heat, acid and oxidative stresses seen in other organisms (Kelly et al. 2001), although there is a noticeable increase in pressure resistance (Martínez-Rodriguez and Mackey 2005). During extended incubation in stationary phase, resistance to pressure fluctuates, as previously found with resistance to heat and oxidative stress (Kelly et al. 2001; Martínez-Rodriguez et al. 2004). From the practical point of view, it is clear that age of culture has a significant effect on pressure resistance that should be borne in mind when harvesting cells for challenge tests.

The survival of resistant and sensitive strains of C. jejuni was only slightly affected by the composition of the suspending medium when tested at 200 MPa but at 300 MPa survival of both strains in milk was much higher than in BBFBP broth. In the case of the weaker strain, survival in the two media differed by nearly 6 log units. The special baroprotective effect of milk seen in other species of bacteria (Patterson et al. 1995; Hauben et al. 1998) thus also applies to C. jejuni, but milk was unable to prevent loss of viability at 400 MPa even in the more resistant strain of C. jejuni.

Solomon and Hoover (2004) found that pressure treatment at 300–325 MPa for 10 min was sufficient to reduce viable numbers of C. jejuni by 8 log units or more in broth or phosphate buffer, whereas in milk or chicken puree the same treatment gave only a 2–3 log decrease. The authors concluded, however, that a pressure treatment at 400 MPa was sufficient to inactivate C. jejuni in food. In the present work treatment of C. jejuni in milk at 300 MPa produced only about 1 log unit reduction but treatment at 400 MPa was sufficient to overcome the baroprotective effect, in agreement with Solomon and Hoover (2004). The risks of Campylobacter sp. surviving commercial pressure treatments are, therefore, very small, even with the more resistant strains in food products that afford substantial protection against pressure.

This work has shown that Campylobacter spp. are sensitive to pressure as they are to most other stresses, yet they are widely dispersed in food and the environment and can be recovered even from apparently hostile conditions such as the sand of bathing beaches provided suitable detection methods are employed (Bolton et al. 1999). The reasons why such apparently fragile organisms are able to survive so well are not entirely understood but despite their ubiquity they do not appear to pose a particular problem for novel food preservation technologies such as high pressure processing.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by a Marie Curie Postdoctoral Fellowship grant to Adolfo Martínez-Rodriguez, from the EC Framework 5 programme, Quality of Life and Management of Living Resources, project no. QLK1-CT-200-52125. We are very grateful to Bernadette Klotz for statistical analyses.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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