Heat Resistance and Mechanism of Heat Inactivation in Thermophilic Campylobacters

The heat resistance of Campylobacter jejuni strains AR6 and L51 and the heat resistance of Campylobacter coli strains DR4 and L6 were measured over the temperature range from 50 to 60°C by two methods. Isothermal measurements yielded D₅₅ values in the range from 4.6 to 6.6 min and z values in the range from 5.5 to 6.3°C. Dynamic measurements using differential scanning calorimetry (DSC) during heating at a rate of 10°C/min yielded D₅₅ values of 2.5 min and 3.4 min and z values of 6.3°C and 6.5°C for AR6 and DR4, respectively. Both dynamic and isothermal methods yielded mean D₅₅ values that were substantially greater than those reported previously (0.75 to 0.95 min). DSC analysis of each strain during heating at a rate of 10°C/min yielded a complex series of overlapping endothermic peaks, which were assigned to cell wall lipids, ribosomes, and DNA. Measurement of the decline in the numbers of CFU in calorimetric samples as they were heated showed that the maximum rate of cell death occurred at 56 to 57°C, which is close to the value predicted mathematically from the isothermal measurements of D and z (61°C). Both estimates were very close to the peak m₁ values, 60 to 62°C, which were tentatively identified with unfolding of the 30S ribosome subunit, showing that cell death in C. jejuni and C. coli coincided with unfolding of the most thermally labile regions of the ribosome. Other measurements indicated that several essential proteins, including the α and β subunits of RNA polymerase, might also unfold at the same time and contribute to cell death.     

Heat resistance of Campylobacter and Yersinia strains by three methods

Two methods of determining the heat resistance of bacteria, a glass cup and a test tube method, were compared with a method using capillary tubes. Three strains of Yersinia enterocolitica , one of Campylobacter jejuni and two of C. coli were tested in physiological saline. The differences between the results obtained by the glass cup method and the reference method were not statistically significant for five strains and were small also for the other, a Yersinia strain. D values obtained by the glass cup method at 58, 60 and 62°C were 1.4–1.8, 0.40–0.51 and 0.15–0.19 min ( z values 4.00–4.52°C) for the Yersinia strains, and 0.42, 0.13 and 0.07 min ( z value 5.07°C) for one C. coli strain. For the other Campylobacter strains, D values of 0.71–0.78, 0.24–0.28 and 0.12–0.14 min ( z values 4.94 and 5.60°C) were recorded at 56, 58 and 60°C. D values obtained at 60°C by the test tube method were 2.7–5.0 min and were considered to be unrealistic.     

Heat resistance of Campylobacter and Yersinia strains by three methods

Two methods of determining the heat resistance of bacteria, a glass cup and a test tube method, were compared with a method using capillary tubes. Three strains of Yersinia enterocolitica, one of Campylobacter jejuni and two of C. coli were tested in physiological saline. The differences between the results obtained by the glass cup method and the reference method were not statistically significant for five strains and were small also for the other, a Yersinia strain. D values obtained by the glass cup method at 58, 60 and 62 degrees C were 1.4-1.8, 0.40-0.51 and 0.15-0.19 min (zeta values 4.00-4.52 degrees C) for the Yersinia strains, and 0.42, 0.13 and 0.07 min (zeta value 5.07 degrees C) for one C. coli strain. For the other Campylobacter strains, D values of 0.71-0.78, 0.24-0.28 and 0.12-0.14 min (zeta values 4.94 and 5.60 degrees C) were recorded at 56, 58 and 60 degrees C. D values obtained at 60 degrees C by the test tube method were 2.7-5.0 min and were considered to be unrealistic.     

Response of Campylobacter jejuni to sodium chloride.

Studies were done to provide more comprehensive information on the response of Campylobacter jejuni and nalidixic acid-resistant, thermophilic Campylobacter (NARTC) to sodium chloride at 4, 25, and 42 degrees C. Three strains of C. jejuni were studies, and all could grow at 42 degrees C in the presence of 1.5% NaCl, but not 2.0% NaCl. At the same temperature, NARTC could grow in 2.0% NaCl and was substantially more tolerant to 2.5 and 4.5% NaCl than was C. jejuni. Both C. jejuni and NARTC grew poorly in the absence of added NaCl and grew best in the presence of 0.5% NaCl at 42 degrees C. At 25 degrees C, NaCl concentrations of 1.0 to 2.5% were protective to NARTC, but the same concentrations of salt generally enhanced the rate of death of C. jejuni. At 4 degrees C, both C. jejuni and NARTC were sensitive to 1.0% or more NaCl; however, the rate of death at this temperature was substantially less than that which occurred at 25 degrees C. A 3 log10 decrease of cells occurred in 4.5% NaCl after 1.2 to 2.1 days at 25 degrees C, and a similar reduction in cells took approximately 2 weeks at the same salt concentration and 4 degrees C. Although C. jejuni grows best in the presence of 0.5% NaCl, the presence of NaCl at concentrations as low as 1.0% may retard growth or increase rate of death; hence, it is advisable that growth media used for recovering or enumerating this organism contain 0.5% NaCl, but not 1.0% or more NaCl.     

The effects of UVB and temperature on the survival of natural populations and pure cultures of Campylobacter jejuni, Camp. coli, Camp. lari and urease-positive thermophilic campylobacters (UPTC) in surface waters

AIMS: To determine whether diurnal and seasonal variations in campylobacters in surface waters result from the effects of temperature and u.v. radiation, and whether natural populations of Campylobacter lari and urease-positive thermophilic campylobacters (UPTC) from birds survive better in surface waters than Camp. jejuni from sewage. METHODS AND RESULTS: Natural populations of Camp. lari and UPTC in sea water, and Camp. jejuni in river water, were exposed to artificial sunlight (equivalent to a sunny day in June). Both populations became non-culturable within 30 min, with T90s of 15 min and 25 min, respectively. Cultures of Camp. jejuni became non-culturable within 40 min and those of Camp. coli, Camp. lari and UPTC, within 60 min. In darkness, survival was temperature-dependent. Natural populations took 12 h at 37 degrees C and 5 days at 4 degrees C to become non-culturable in sea water, and slightly less in river water. Cultures of Camp. lari and UPTCs survived for significantly longer than Camp. jejuni and Camp. coli. Loss of culturability for all isolates was most rapid at 37 degrees C and slowest at 4 degrees C. Newly isolated strains from sea water and river water behaved in an almost identical manner to NCTC strains. CONCLUSION: Campylobacter lari and UPTCs survive for longer in surface waters than Camp. jejuni and Camp. coli, particularly in the dark. Low Campylobacter numbers in coastal waters in the summer, especially in the afternoon, are due to the combined effects of higher temperatures and higher levels of u.v. radiation. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter lari and UPTCs from birds predominate in bathing waters in Morecambe Bay because they are better able to survive; they also originate from closer to the shore than Camp. jejuni and Camp. coli in sewage effluent, which survive poorly and die before the incoming tide reaches the shore. The predominance of Camp. jejuni in river water results from its dominance of the inputs and not from its ability to survive.     

Campylobacter jejuni survival in chicken meat as a function of temperature.

Recognition of Campylobacter fetus subsp. jejuni (referred to hereafter as C. jejuni) as an important human pathogen and its isolation from meat products indicate the need for knowledge of its survival characteristics in meats. Thermal death times (D-values) for a single strain and a five-strain composite were determined in 1% peptone and autoclaved ground chicken meat at temperatures ranging from 49 to 57 degrees C. Survival was determined for these strains in chicken meat at 4, 23, 37, and 43 degrees C. Survival was also determined on raw chicken drumsticks stored at 4 degrees C in either an ambient or a CO2 atmosphere. D-values were greater in chicken meat than in peptone in all cases. D-values in peptone for strain H-840 at 49, 51, 53, 55, and 57 degrees C were 15.2, 4.90, 1.71, 0,64, and 0.25 min, respectively. The corresponding D-values in ground chicken meat were 20.5, 8.77, 4.85, 2.12, and 0.79 min, respectively. Similar results were obtained with a composite of five strains. When sterile ground chicken meat was inoculated with approximately 10(6) to 10(7) C. jejuni cells per g and stored at 37 degrees C in an ambient atmosphere, a 1-to 2-log count increase occurred during the first 4 days, followed by a gradual decline of about 1 log during the remainder of the 17-day storage period. No growth was observed among similarly inoculated samples that were stored at 4, 23, and 43 degrees C but counts declined by about 1 to 2 logs at 4 degrees C (17 day), by 2.5 to 5 logs at 23 degrees C (17 days), and to undetectable levels at 43 degrees C (between 10 and 16 days). Survival on raw chicken drumsticks stored at 4 degrees C in CO2 and in an ambient atmosphere declined by about 1.5 and 2.0 logs, respectively, during 21 days of storage. The effect of temperature on the survival of C. jejuni in chicken meat was similar to that reported in other natural and laboratory milieus. Ordinary cooking procedures that destroy salmonellae would be expected to destroy C. jejuni.     

Effect of incubation temperature on isolation of Campylobacter jejuni genotypes from foodstuffs enriched in Preston broth

Preston broth and agar incubated at either 37 or 42 degrees C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42 degrees C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37 degrees C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37 degrees C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42 degrees C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42 degrees C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.     

The effect of thermal stress on Campylobacter coli

AIM: Enteropathogenic Campylobacter jejuni, Camp. coli and Camp. lari are currently the most common cause of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs for these organisms and infection may occur through the ingestion of contaminated foodstuffs. The safety of locally produced porcine liver was assessed in relation to the heat susceptibility of Campylobacter spp. present in eviscerated product. METHODS AND RESULTS: Heat susceptibility (D10) studies were performed on a wild-type strain of Camp. coli [NI39] isolated from porcine liver under standardized conditions. In addition, the effect of culture age and heating menstruum was determined. Thermal stress studies in phosphate-buffered saline showed Camp. coli NI39 to be heat sensitive (D10 = 8.-0, 30.8, 15.6, 10.3 s at 55.4, 57.4, 59.7, 61.2 degrees C, respectively; z = 6.10 degrees C). However, non-logarithmic biphasic survivor curves were observed at higher temperatures ( > 56 degrees C), indicating the presence of a heat-resistant subpopulation (10(4)-10(5) cfu) which was not demonstrated when examining either Salmonella typhimurium or Listeria monocytogenes. CONCLUSIONS: The use of D10 values may be limited. Therefore, porcine liver, under processing, must be treated as a potential source of Campylobacter spp., and clearly defined F-values should be quantified through the use of empirically 'spiked' samples to ensure the eradication of campylobacters from the product, for each individual process being evaluated. SIGNIFICANCE AND IMPACT OF THE STUDY: It is important to define safe processing parameters in the manufacture of products which receive mild thermal processes in order to eliminate the risk of disease to man.     

PCR-ELISAs for the detection of Campylobacter jejuni and Campylobacter coli in poultry samples

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are regarded as a major cause of human gastrointestinal disease, commonly acquired by eating undercooked chicken. We describe a PCR-ELISA for the detection of Campylobacter species and the discrimination of C. jejuni and C. coli in poultry samples. The PCR assay targets the 16S/23S ribosomal RNA intergenic spacer region of Campylobacter species with DNA oligonucleotide probes designed for the specific detection of C. jejuni, C. coli, and Campylobacter species immobilized on Nucleo-Link wells and hybridized to PCR products modified with a 5' biotin moiety. The limit of detection of the PCR-ELISA was 100-300 fg (40-120 bacterial cells) for C. jejuni and C. coli with their respective species-specific oligonucleotide probes and 10 fg (4 bacterial cells) with the Campylobacter genus-specific probe. Testing of poultry samples, which were presumptive positive for Campylobacter following culture on the Malthus V analyzer, with the PCR-ELISA determined Campylobacter to be present in 100% of samples (n = 40) with mixed cultures of C. jejuni/C. coli in 55%. The PCR-ELISA when combined with culture pre-enrichment is able to detect the presence of Campylobacter and definitively identify C. jejuni and C. coli in culture-enriched poultry meat samples.     

Efficacy of media and methods for detecting and enumerating Campylobacter jejuni in refrigerated chicken meat.

A study was undertaken to compare several enrichment and direct isolation media for their suitability to detect and enumerate five strains of Campylobacter jejuni in refrigerated (5 degrees C) chicken meat. The influence of CO2 on survival at 5 degrees C was also investigated. Selective enrichment media evaluated included Preston broth (PB), selective semisolid brucella medium (SSBM), Campylobacter enrichment broth (CEB), VTP brucella-FBP broth (VTP), Rosef and Kapperud Campylobacter enrichment broth (RKCEB), and Doyle and Roman enrichment broth (DREB). Direct isolation agars included Campy brucella agar (CBAP), blood-free Campylobacter medium (BFCM) and modified Butzler agar (MBA). Comminuted chicken meat was inoculated with C. jejuni, sealed under atmospheric gas or CO2, and stored at 5 degrees C for up to 21 days. Viable population was determined by the most-probable-number technique (PB, SSBM, CEB, VTP, and RKCEB, followed by plating on CBAP, BFCM, and MBA), enrichment on DREB, followed by plating on CBAP, BFCM, and MBA, and direct isolation on CBAP, BFCM, and MBA. Without exception, direct plating of samples was superior to the most-probable-number technique for enumerating C. jejuni; MBA was inferior to CBAP and BFCM, and DREB performed at least as well as other enrichment media evaluated. Carbon dioxide afforded protection against death of three of the five strains of C. jejuni tested.     

Efficacy of media and methods for detecting and enumerating Campylobacter jejuni in refrigerated chicken meat

A study was undertaken to compare several enrichment and direct isolation media for their suitability to detect and enumerate five strains of Campylobacter jejuni in refrigerated (5 degrees C) chicken meat. The influence of CO2 on survival at 5 degrees C was also investigated. Selective enrichment media evaluated included Preston broth (PB), selective semisolid brucella medium (SSBM), Campylobacter enrichment broth (CEB), VTP brucella-FBP broth (VTP), Rosef and Kapperud Campylobacter enrichment broth (RKCEB), and Doyle and Roman enrichment broth (DREB). Direct isolation agars included Campy brucella agar (CBAP), blood-free Campylobacter medium (BFCM) and modified Butzler agar (MBA). Comminuted chicken meat was inoculated with C. jejuni, sealed under atmospheric gas or CO2, and stored at 5 degrees C for up to 21 days. Viable population was determined by the most-probable-number technique (PB, SSBM, CEB, VTP, and RKCEB, followed by plating on CBAP, BFCM, and MBA), enrichment on DREB, followed by plating on CBAP, BFCM, and MBA, and direct isolation on CBAP, BFCM, and MBA. Without exception, direct plating of samples was superior to the most-probable-number technique for enumerating C. jejuni; MBA was inferior to CBAP and BFCM, and DREB performed at least as well as other enrichment media evaluated. Carbon dioxide afforded protection against death of three of the five strains of C. jejuni tested.     

Inactivation of Campylobacter jejuni by high hydrostatic pressure

AIMS: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. METHODS AND RESULTS: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25 degrees C suspended in Bolton broth, phosphate buffer (0.2 m, pH 7.3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300-325 MPa for 10 min at 25 degrees C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50-75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: These data demonstrated that HPP at levels of 相似文献   

Survival of Campylobacter jejuni inoculated into ground beef.

Ground beef was inoculated with mixed cultures of Campylobacter jejuni, and the samples were subjected to various cooking and cold-storage temperatures. When samples were heated in an oven at either 190 or 218 degrees C, approximately 10(7) cells of C. jejuni per g were inactivated (less than 30 cells per g) in less than 10 min after the ground beef reached an internal temperature of 70 degrees C. When the samples were held at -15 degrees C over 14 days of storage, the numbers of C. jejuni declined by 3 log10. When inoculated samples were stored with an equal amount of Cary-Blair diluent at 4 degrees C, no changes in viability were observed over 14 days of storage. Twenty-five times as much C. jejuni was recovered from inoculated ground beef when either 10% glycerol or 10% dimethyl sulfoxide was added to an equal amount of ground beef before freezing as was recovered from peptone-diluted ground beef. Twice as much inoculated C. jejuni was recovered from ground beef plus Cary-Blair diluent as was recovered from ground beef plus peptone diluent.     

Inactivation of Escherichia coli O157:H7, salmonellae, and Campylobacter jejuni in raw ground beef by gamma irradiation.

Raw ground beef patties inoculated with stationary-phase cells of Escherichia coli O157:H7, salmonellae, or Campylobacter jejuni were subjected to gamma irradiation (60Co) treatment, with doses ranging from 0 to 2.52 kGy. The influence of two levels of fat (8 to 14% [low fat] and 27 to 28% [high fat]) and temperature (frozen [-17 to -15 degrees C] and refrigerated [3 to 5 degrees C]) on the inactivation of each pathogen by irradiation was investigated. In ascending order of irradiation resistance, the D10 values ranged from 0.175 to 0.235 kGy (C. jejuni), from 0.241 to 0.307 kGy (E. coli O157:H7), and from 0.618 to 0.800 kGy (salmonellae). Statistical analysis revealed that E. coli O157:H7 had a significantly (P < 0.05) higher D10 value when irradiated at -17 to -15 degrees C than when irradiated at 3 to 5 degrees C. Regardless of the temperature during irradiation, the level of fat did not have a significant effect on the D10 value. Salmonellae behaved like E. coli O157:H7 in low-fat beef, but temperature did not have a significant effect when the pathogen was irradiated in high-fat ground beef. Significantly higher D10 values were calculated for C. jejuni irradiated in frozen than in refrigerated low-fat beef. C. jejuni was more resistant to irradiation in low-fat beef than in high-fat beef when treatment was at -17 to -15 degrees C. Regardless of the fat level and temperature during inactivation, these pathogens were highly sensitive to gamma irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the survival of Campylobacter jejuni and Campylobacter coli in culturable form in surface water

Six Campylobacter jejuni and six Campylobacter coli strains were isolated from cows and pigs, and their survival in lake water was compared by viable counts. Campylobacter jejuni survived longer in culturable form than C. coli in untreated and membrane-filtered water both at 4 and 20 degrees C. This difference in survival time may be a reason why C. jejuni is generally isolated from surface waters more frequently than C. coli. Both species survived better in filtered than in untreated water. This suggests that predation and competition for nutrients affect the survival of both Campylobacter species in the aquatic environment.     

Effect of temperature on growth and chemotactic behaviour of Campylobacter jejuni

AIM: To determine the effect of two physiologically important temperatures on growth and chemotaxis in Campylobacter jejuni. METHODS AND RESULTS: Growth curves of Camp. jejuni were compared at 37 degrees C and 42 degrees C. Chemotaxis was compared at 37 degrees C and 42 degrees C by the disc and capillary assays. Student's t-test was applied to the results of the capillary assay to assess the significance in the difference between chemotaxis at the two temperatures. Both, the growth rate and chemotactic ability of the isolate, were found to be greater at 37 degrees C. CONCLUSIONS: Quorum sensing (related to population density), a regulation mechanism of virulence in micro-organisms, has been reported in Campylobacter. Chemotaxis is also a known virulence factor of Campylobacter. Both, growth (in terms of population density) and chemotaxis, being greater at 37 degrees C than at 42 degrees C, suggests that the physiological temperature of humans (37 degrees C) might be more favourable for the expression of virulence in Campylobacter than that of birds (42 degrees C). SIGNIFICANCE AND IMPACT OF THE STUDY: It is as yet not known why Campylobacter causes disease in humans but is avirulent in birds. This study suggests that the human body temperature is optimum for growth and chemotaxis in Campylobacter. There is scope for the study of temperature regulation of other virulence determinants of Campylobacter.     

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

Volume, expansivity and isothermal compressibility changes associated with temperature and pressure unfolding of Staphylococcal nuclease

We have characterized the temperature- and pressure-induced unfolding of staphylococcal nuclease (Snase) using high precision densitometric measurements. The changes in the apparent specific volume, expansion coefficient and isothermal compressibility were determined by these measurements. To our knowledge, these are the first measurements of the volume and isothermal compressibility changes of a protein undergoing pressure-induced unfolding. In order to aid in interpreting the temperature and pressure dependence of the apparent specific volume of Snase, we have also carried out differential scanning calorimetry under the solution conditions which are used for the volumetric studies. We have seen that large compensating volume and compressibility effects accompany the temperature and pressure-induced protein unfolding. Measurements of the apparent specific volume and thermal expansion coefficient of Snase at ambient pressure indicate the formation of a pre-transitional, molten globule type of intermediate structure about 10 degrees C below the actual unfolding temperature of the protein. Compared to the folded state, the apparent specific volume of the unfolded protein is about 0.3-0.5 % smaller. In addition, we investigated the pressure dependence of the apparent specific volume of Snase at a number of different temperatures. At 45 degrees C we calculate a decrease in apparent specific volume due to pressure-induced unfolding of -3.3 10(-3) cm(3) g(-1) or -55 cm(3) mol(-1). The threefold increase in compressibility between 40 and 70 MPa reflects a transition to a partially unfolded state, which is consistent with our results obtained for the radius of gyration of the pressure-denatured state of Snase. At the lower temperature of 35 degrees C, a significant increase in compressibility around 30 MPa is indicative of the formation of a pressure-induced molten globule-like intermediate. Changes in the apparent volume, expansion coefficient and isothermal compressibility are discussed in terms of instrinsic, hydrational and thermal contributions accompanying the unfolding transition.     

Campylobacter jejuni survival within human epithelial cells is enhanced by the secreted protein CiaI

Although it is known that Campylobacter jejuni invade the cells that line the human intestinal tract, the bacterial proteins that enable this pathogen to survive within Campylobacter-containing vacuoles (CCV) have not been identified. Here, we describe the identification and characterization of a protein that we termed CiaI for Campylobacter invasion antigen involved in intracellular survival. We show that CiaI harbours an amino-terminal type III secretion sequence and is secreted from C. jejuni through the flagellar type III secretion system. In addition, the ciaI mutant was impaired in intracellular survival when compared with a wild-type strain, as judged by the gentamicin-protection assay. Fluorescence microscopy examination of epithelial cells infected with the C. jejuni ciaI mutant revealed that the CCV were more frequently co-localized with Cathepsin D (a lysosomal marker) than the CCV in cells infected with a C. jejuni wild-type strain. Ectopic expression of CiaI-GFP in epithelial cells yielded a punctate phenotype not observed with the other C. jejuni genes, and this phenotype was abolished by mutation of a dileucine motif located in the carboxy-terminus of the protein. Based on the data, we conclude that CiaI contributes to the ability of C. jejuni to survive within epithelial cells.