The Effect of Nacl on Campylobacter Jejuni/Coli

The growth of 2 strains of Campylobacter jejuni/coli was investigated in 0–2.0 % NaCl in Brucella broth at 35° G and 30° C. Both strains tolerated more NaCl in the growth medium at 35° C than at 30° C. 2 % NaCl was bacteriocidic at both temperatures. The strains also grew in the medium without added NaCl. At 35° C, low concentrations of NaCl stimulated the growth of strain 5616, but not the growth of strain B33. At 30° C, strain 5616 grew in NaCl concentrations up to 1.0 % and strain B33 in 0 % and at the control concentration (0.5 % NaCl). The survival of 22 C. jejuni/coli strains in 2.0 % NaCl at 4° C and 35° C was also investigated. Human strains showed significantly greater tolerance to 2.0 % NaCl at both temperatures than did the strains isolated from animals. These findings suggest that the salting of food can be effective in preventing the growth or survival of C. jejuni/coli.     

Differential characteristics of catalase-positive campylobacters correlated with DNA homology groups

Eighty-four strains of catalase-positive campylobacters could be placed into seven distinct DNA homology groups (species), corresponding to Campylobacter fetus, "C. hyointestinalis," C. jejuni, C. coli, "C. laridis," "C. fecalis," and aerotolerant campylobacters. The biochemical and physiological characteristics of the strains were examined for their correlation with the homology groups. The characterization tests that provided the most reliable differentiation at the species and subspecies level were growth at 25 and 42 degrees C, sensitivity to cephalothin and nalidixic acid, growth in semisolid media containing 1% glycine and 3.5% NaCl, growth on plates containing 1.5% NaCl, growth in a semisolid minimal medium, anaerobic growth in the presence of 0.1% trimethylamine-N-oxide, hydrogen sulfide production in SIM medium and triple-sugar iron agar, hippurate hydrolysis, nitrite reduction, and growth on plates under an air atmosphere.     

Survival and growth of Campylobacter fetus subsp. jejuni on meat and in cooked foods.

Twelve strains of Campylobacter fetus subsp. jejuni isolated from humans and animals grew at temperatures ranging from 34 to 45 degrees C and pH minima between 5.7 and 5.9. Only one strain grew at pH 5.8 with lactic acid present at a concentration similar to that in meat. All strains had decimal reduction times of less than 1 min at 60 degrees C. Further examination of a typical strain showed that it grew at 37 degrees C on high-pH meat but not at 37 degrees C on normal-pH meat. Bacterial numbers on both high (6.4)-pH and normal (5.8)-pH inoculated meat declined at a similar rate when the meat was stored at 25 degrees C. At -1 degree C, the rate of die-off was somewhat slower on normal-pH meat but was very much slower on high-pH meat. The initial fall in bacterial numbers that occurred when meat was frozen was also greater for normal-pH meat than for high-pH meat. The organism exhibited a long lag phase (1 to 2 days) when grown in cooked-meat medium at 37 degrees C and died in meat pies stored at 37 or 43 degrees C. Evaluation of the risk of Campylobacter contamination of red-meat carcasses to human health must take into account the limited potential of the organism to grow or even survive on fresh meats and in warm prepared foods.     

Effect of incubation atmosphere and temperature on isolation of Campylobacter jejuni from human stools

To determine the optimal conditions for isolation of Campylobacter jejuni from human fecal specimens, we compared incubation atmospheres that contained about 5, 10, and 15% oxygen with the 17% oxygen produced in candle jars and also compared incubation temperatures of 37 and 42 degrees C. At 42 degrees C, C. jejuni was isolated from all 16 specimens; however, colony sizes were larger when plates were incubated in 5 and 10% oxygen than in the other two atmospheres. At 37 degrees C some positive cultures were missed in 15% oxygen and in the candle jar. The largest colony sizes were obtained in 5% oxygen. For each atmospheric condition tested, the colonies were larger at 42 than at 37 degrees C. When incubation is done at 42 degrees C, use of a candle jar is adequate; however, at 37 degrees C candle jars should not be used for isolation of C. jejuni from human feces.     

Study of haemolytic activity of some Campylobacter spp. on blood agar plates

A total of 152 strains of Campylobacter jejuni, C. coli, C. laridis and C. fetus subsp. fetus were tested for haemolysis on blood agar plates. Distinct haemolysis was detected in 92.3% (96/104) of strains of C. jejuni and 21.7% (5/23) of strains of C. coli on sheep blood heart infusion agar after incubation for 4 d microaerobically at 42 degrees C. Haemolysis was also detected on horse blood heart infusion agar. Haemolysis was not detected at 37 degrees C except with one of 50 strains of C. jejuni tested at this temperature, which was weakly positive. Campylobacter laridis was not haemolytic; C. fetus subsp. fetus, which does not grow at 42 degrees C, showed no haemolysis at 37 degrees C. Blood agar (Oxoid, BA Base No. 2) was not suitable for testing for haemolysis by these organisms. A microaerobic gas mixture containing hydrogen is better than that containing nitrogen because the medium has a brighter colour, making haemolysis easier to detect. There was no synergistic haemolysis with Staphylococcus aureus or Streptococcus agalactiae. The plate haemolysis test as described here may aid differentiation within the thermophilic campylobacters.     

Heat injury and repair in Campylobacter jejuni

A procedure for detecting and quantitating heat injury in Campylobacter jejuni was developed. Washed cells of C. jejuni A7455 were heated in potassium phosphate buffer (0.1 M, pH 7.3) at 46 degrees C. Samples were plated on brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate and on a medium containing brilliant green, bile, Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate. Colonies were counted after 5 days of incubation at 37 degrees C in an atmosphere containing 5% O2, 10% CO2, and 85% N2. After 45 min at 46 degrees C, there was virtually no killing and ca. two log cycles of injury. Cells grown at 42 degrees C were more susceptible to injury than cells grown at 37 degrees C. The addition to brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate of three different antibiotic mixtures used in the isolation of C. jejuni from foods or clinical specimens did not prevent recovery of heat-injured C. jejuni. Cells lost 260 nm of absorbing materials during heat injury. The addition of 5% NaCl or 40% sucrose to the heating buffer prevented leakage but did not prevent injury. Of the additional salts, sugars, and amino acids tested for protection, only NH4Cl, KCl, and LiCl2 prevented injury. Heat-injured C. jejuni repaired (regained dye and bile tolerance) in brucella broth supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate within 4 h. Increasing the NaCl in this medium to 1.25% inhibited repair, and increasing it to 2% was lethal. Heat-injured C. jejuni will repair at 42 degrees C but not at 5 degrees C.     

Heat injury and repair in Campylobacter jejuni.

A procedure for detecting and quantitating heat injury in Campylobacter jejuni was developed. Washed cells of C. jejuni A7455 were heated in potassium phosphate buffer (0.1 M, pH 7.3) at 46 degrees C. Samples were plated on brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate and on a medium containing brilliant green, bile, Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate. Colonies were counted after 5 days of incubation at 37 degrees C in an atmosphere containing 5% O2, 10% CO2, and 85% N2. After 45 min at 46 degrees C, there was virtually no killing and ca. two log cycles of injury. Cells grown at 42 degrees C were more susceptible to injury than cells grown at 37 degrees C. The addition to brucella agar supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate of three different antibiotic mixtures used in the isolation of C. jejuni from foods or clinical specimens did not prevent recovery of heat-injured C. jejuni. Cells lost 260 nm of absorbing materials during heat injury. The addition of 5% NaCl or 40% sucrose to the heating buffer prevented leakage but did not prevent injury. Of the additional salts, sugars, and amino acids tested for protection, only NH4Cl, KCl, and LiCl2 prevented injury. Heat-injured C. jejuni repaired (regained dye and bile tolerance) in brucella broth supplemented with Na2S2O3, FeSO4 X 7H2O, and sodium pyruvate within 4 h. Increasing the NaCl in this medium to 1.25% inhibited repair, and increasing it to 2% was lethal. Heat-injured C. jejuni will repair at 42 degrees C but not at 5 degrees C.     

Behaviour of Campylobacter jejuni in experimentally contaminated bottled natural mineral water

AIMS: The main objective of the present study was to estimate the survival of microaerophilic Campylobacter jejuni in filtered natural mineral water at 4 degrees C and 25 degrees C. The influence of the presence of biodegradable organic matter was tested, assuming that the bacterial contamination of a bottled natural mineral water could be associated with contamination by organic matter. METHODS AND RESULTS: Washed Campylobacter cultures were inoculated in natural mineral water and sterile natural mineral water, and incubated in the dark at 4 degrees C and 25 degrees C. The effect of temperature, the biodegradable organic matter added, incubation atmosphere and autochthonous microflora were tested on the cultivability of Camp. jejuni. CONCLUSIONS: The survival of Camp. jejuni in natural mineral water was better at 4 degrees C than at 25 degrees C, and the presence of organic matter led to a deceleration in the loss of cultivability and to the multiplication of Camp. jejuni in natural mineral water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that, in the event of dual contamination of a bottled natural mineral water (Campylobacter and biodegradable organic matter), the pathogen could survive (and even grow) for a relatively long time, especially at low temperature and in spite of the presence of oxygen.     

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.     

Differential effects of temperature on natural transformation to erythromycin and nalidixic acid resistance in Campylobacter coli

Campylobacter jejuni and Campylobacter coli are naturally competent, but limited information exists on the impact of environmental conditions on transformation. In this study, we investigated the impact of temperature and microaerobic versus aerobic atmosphere on transformation of C. coli to erythromycin and nalidixic acid resistance. Frequency of transformation was not significantly different between microaerobic (5 to 10% CO(2)) and aerobic conditions. However, C. coli was transformed to erythromycin resistance at a significantly higher frequency at 42 degrees C than at 25 degrees C (P < 0.05), and few or no transformants were obtained at 25 degrees C. In contrast, transformation to nalidixic acid resistance was highly efficient at both 42 degrees C and 25 degrees C and was similar or, at the most, fourfold higher at 42 degrees C than at 25 degrees C. DNase I treatment experiments suggested that steps both prior and subsequent to internalization of DNA were influenced by temperature in the case of transformation of C. coli to erythromycin resistance. However, the moderately increased (fourfold) frequency of transformation to nalidixic acid resistance at 42 degrees C compared to that at 25 degrees C was exclusively associated with steps prior to DNA internalization. These findings suggest that transformation to erythromycin resistance may be significantly more frequent in the gastrointestinal tract of hosts such as poultry (at 42 degrees C) than in other habitats characterized by lower temperatures, whereas transformation to nalidixic acid resistance may be highly efficient both within and outside the animal hosts.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni. The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO2 + 80% N2, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O2 + 10% CO2 + 85% N2. The packaging material in the first two treatments was PA 80/PE 100-PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37 degrees C, 20 degrees C and 4 degrees C for 48 h, 4 days and 25 days, respectively. At 37 degrees C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20 degrees C and at 4 degrees C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37 degrees C its numbers increased only in the optimal gas atmosphere; at 20 degrees C the strain was not detectable after 24 to 48 h storage and at 4 degrees C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Impact of different storage factors on the survivability of Campylobacter jejuni in turkey meat

Campylobacter jejuni is often prevalent in turkey and poultry, but the effects of storage temperatures and storage periods and the interruption of the cooling chain on its survival have not been evaluated so far. In this study, 700 samples of turkey meat were artificially contaminated by inoculating their surface with 10(3) CFU of C. jejuni per sample, wrapped in airtight cellophane bags, and stored under different chilling and freezing conditions for various storage periods; this was followed by analysis of the cultures. Subsequent to incubation at 25 degrees C for 48 h, C. jejuni was reisolated in only 7% of the samples. When the samples were stored under refrigerator conditions at 4 degrees C, the organism was reisolated in 42% of the samples after 1 week, and in 28% of the samples after 2 weeks. The recovery rates in the samples that had been stored frozen at -20 degrees C without interruption of the cooling chain were 68% after 2 weeks and 24% after 4 weeks. Different storage conditions were simulated in order to examine the impact of an interruption of the cooling chain on the survival of Campylobacter.     

A novel method and simple apparatus for the detection of thermophilic Campylobacter spp. in chicken meat products

Conventional procedures for isolation of thermophilic Campylobacter spp. from chicken are complex, labor intensive, and time-consuming. The objective of this study was to create a novel Campylobacter culturing apparatus. A main concept of the device was based on the ability of Campylobacter to pass through a 0.45 microm pore size filter in viscous media. Preliminary study demonstrated that only viable Campylobacter moved through the membrane filter and could multiply in the enrichment culture. C. jejuni, C. coli, C. lari, and C. upsaliensis in the chicken samples were detected at cell concentrations as low as 10 cfu/g, after 24 h incubation at 42 degrees C. In total, 84 retail chicken samples were comparatively studied using both conventional method and apparatus. Sixteen samples (19.05%) were positive by the apparatus method; 14 (16.66%) of these positive samples contained C. coli and 2 (2.38%) contained C. jejuni. With the conventional method, 7 (8.33%) samples were positive 7 (8.33%) with C. coli. In conclusion, the apparatus detected more positive samples than did the conventional culture method.     

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.     

Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice

Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Inactivation of Campylobacter jejuni by chlorine and monochloramine.

Campylobacter jejuni and closely related organisms are important bacterial causes of acute diarrheal illness in the United States. Both endemic and epidemic infections have been associated with consuming untreated or improperly treated surface water. We compared susceptibility of three C. jejuni strains and Escherichia coli ATCC 11229 with standard procedures used to disinfect water. Inactivation of bacterial preparations with 0.1 mg of chlorine and 1.0 mg of monochloramine per liter was determined at pH 6 and 8 and at 4 and 25 degrees C. Under virtually every condition tested, each of the three C. jejuni strains was more susceptible than the E. coli control strain, with greater than 99% inactivation after 15 min of contact with 1.0 mg of monochloramine per liter or 5 min of contact with 0.1 mg of free chlorine per liter. Results of experiments in which an antibiotic-containing medium was used suggest that a high proportion of the remaining cells were injured. An animal-passaged C. jejuni strain was as susceptible to chlorine disinfection as were laboratory-passaged strains. These results suggest that disinfection procedures commonly used for treatment of drinking water to remove coliform bacteria are adequate to eliminate C. jejuni and further correlate with the absence of outbreaks associated with properly treated water.     

Campylobacter jejuni inactivation in New Zealand soils

AIM: The study was undertaken to determine the inactivation rate of Campylobacter jejuni in New Zealand soils. METHODS AND RESULTS: Farm dairy effluent (FDE) inoculated at c. 10(5) ml(-1) with C. jejuni was applied to intact soil cores at a rate of 2 l m(-2). Four soils were used: Hamilton (granular); Taupo (pumice); Horotiu and Waihou (allophanic). After FDE application cores were incubated at 10 degrees C for up to 32 days. For all four soils all the FDE remained within the cores and at least 99% of C. jejuni were retained in the top 5 cm. Campylobacter jejuni had declined to the limit of detection (two C. jejuni 100 g(-1)) by 25 days in Hamilton and Taupo soils and by 32 days in Waihou soil. In contrast, in Horotiu soil the decline was only three orders of magnitude after 32 days. Simulated heavy rainfall was applied 4 and 11 days after FDE application and only about 1% of the applied C. jejuni were recovered in leachates. CONCLUSIONS: This study demonstrated that at least 99% of applied C. jejuni were retained in the top 5 cm of four soils where they survived for at least 25 days at 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil retention of C. jejuni is efficient at FDE application rates that prevent drainage losses. The low infectious dose of C. jejuni and its ability to survive up to 25 days have implications for stock management on dairy farms.     

Osmotic Reversal of Temperature Sensitivity in Escherichia coli

Forty temperature-sensitive mutants, unable to grow on tryptone or nutrient agar at 42 C, were isolated from Escherichia coli K-12. When 0.5% NaCl was added to the medium, 32 grew at the nonpermissive temperature. Several were tested with different amounts of NaCl added to tryptone broth; all grew best when the osmolality of the medium was between 400 and 1,000 milliosmolal. One of the mutants was studied in more detail. Sucrose, inositol, KCl, and MgCl(2), as well as NaCl, permitted growth at 42 C. Glycerol, however, had no effect. When shifted from 30 to 42 C without osmotic protection, the mutant stopped growing but did not lyse, die, or leak significant amounts of intracellular material. In a similar shift experiment, a second mutant leaked all of its trichloroacetic acid-soluble pools into the medium. The majority of the mutants were hypersensitive to certain antibiotics, indicating possible cell envelope defects.     

Comparison of survival of Campylobacter jejuni in the phyllosphere with that in the rhizosphere of spinach and radish plants

Campylobacter jejuni has been isolated previously from market produce and has caused gastroenteritis outbreaks linked to produce. We have tested the ability of this human pathogen to utilize organic compounds that are present in leaf and root exudates and to survive in the plant environment under various conditions. Carbon utilization profiles revealed that C. jejuni can utilize many organic acids and amino acids available on leaves and roots. Despite the presence of suitable substrates in the phyllosphere and the rhizosphere, C. jejuni was unable to grow on lettuce and spinach leaves and on spinach and radish roots of plants incubated at 33 degrees C, a temperature that is conducive to its growth in vitro. However, C. jejuni was cultured from radish roots and from the spinach rhizosphere for at least 23 and 28 days, respectively, at 10 degrees C. This enteric pathogen also persisted in the rhizosphere of spinach for prolonged periods of time at 16 degrees C, a temperature at which many cool-season crops are grown. The decline rate constants of C. jejuni populations in the spinach and radish rhizosphere were 10- and 6-fold lower, respectively, than on healthy spinach leaves at 10 degrees C. The enhanced survival of C. jejuni in soil and in the rhizosphere may be a significant factor in its contamination cycle in the environment and may be associated with the sporadic C. jejuni incidence and campylobacteriosis outbreaks linked to produce.