Techniques for the optimum recovery of cold injured Campylobacter jejuni from milk or water

When broth was inoculated with cells of Campylobacter jejuni that had been injured by chilling there was a fall in the viable population of up to 90%. It was greater at 43 degrees than 37 degrees C and in the presence of certain antibiotics and in some cases resulted in a surviving population that was below the minimum inoculum for subsequent growth. A technique of pre-enrichment in non-selective culture broth at 37 degrees C for 2 h before the addition of antibiotics and incubation at 43 degrees C was found to significantly reduce the fall in numbers and to improve the detection of C. jejuni in samples of raw milk and water.     

Techniques for the optimum recovery of cold injured Campylobacter jejuni from milk or water

When broth was inoculated with cells of Campylobacter jejuni that had been injured by chilling there was a fall in the viable population of up to 90%. It was greater at 43° than 37°C and in the presence of certain antibiotics and in some cases resulted in a surviving population that was below the minimum inoculum for subsequent growth. A technique of pre-enrichment in non-selective culture broth at 37°C for 2 h before the addition of antibiotics and incubation at 43°C was found to significantly reduce the fall in numbers and to improve the detection of C. jejuni in samples of raw milk and water.     

An appraisal of the efficacy of pre-enrichment for the isolation of Campylobacter jejuni from water and food

Cells of Campylobacter jejuni exposed to heating or freezing were progressively less able to grow at 43°C, particularly on selective media. This influenced the recovery of damaged cells from naturally and artificially contaminated samples. With broth culture the isolation rate could be increased by pre-enrichment in basal or selective media at 37°C for 4 h. With membrane filtration or surface plating techniques the inclusion of agents that quench toxic derivatives of oxygen was more important.     

An appraisal of the efficacy of pre-enrichment for the isolation of Campylobacter jejuni from water and food

Cells of Campylobacter jejuni exposed to heating or freezing were progressively less able to grow at 43 degrees C, particularly on selective media. This influenced the recovery of damaged cells from naturally and artificially contaminated samples. With broth culture the isolation rate could be increased by pre-enrichment in basal or selective media at 37 degrees C for 4 h. With membrane filtration or surface plating techniques the inclusion of agents that quench toxic derivatives of oxygen was more important.     

Isolation of Campylobacter jejuni from raw milk

Campylobacter jejuni was isolated from raw milk by a method that can routinely detect less than or equal to 1 organism per ml. This procedure was used in a survey of 195 separate farms and showed a 1.5% incidence of C. jejuni in milk from bulk tanks.     

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.     

Isolation of Campylobacter jejuni from raw milk.

Campylobacter jejuni was isolated from raw milk by a method that can routinely detect less than or equal to 1 organism per ml. This procedure was used in a survey of 195 separate farms and showed a 1.5% incidence of C. jejuni in milk from bulk tanks.     

Aerobic growth and survival of Campylobacter jejuni in food and stream water

When 40 Campylobacter jejuni isolates from human clinical cases, raw chicken and water were tested, 29 (72·5%) could be adapted to grow on nutrient agar under aerobic conditions. Once adapted, these isolates could grow on repeated aerobic subculture. An aerobically-grown Camp. jejuni isolate survived almost as well as the same isolate grown microaerophilically in sterile chicken mince at 5 °C, and survival of a cocktail of Camp. jejuni isolates under both atmospheres was comparable at 25 °C. However, at 37 °C, the decline in numbers of the aerobically-grown cells was greater. Survival of cells on chicken nuggets was poorer than in chicken mince. In filter-sterilized stream water incubated aerobically at 5 °C, survival of inocula grown under different atmospheres was again similar, but slightly better with the microaerophilically-grown cells. Adaptation to aerobic growth was not found to enhance survival under aerobic conditions.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.     

Comparison of methods for isolating Campylobacter jejuni from raw milk

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

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.     

Methods for isolating Campylobacter jejuni from low-turbidity water

Membrane filtration methods were developed and evaluated for the quantitative recovery of Campylobacter jejuni from environmental waters of low turbidity. The best procedure studied involved passaging the test water through a filter (pore size, 0.45 micron) and plating it facedown on Campylobacter-selective agar. The filter was removed after overnight incubation, and the plate was streaked for isolation and then reincubated. This method, with or without prefiltration through 5.0- and 0.6-micron-pore-size membranes consistently resulted in the recovery of 30 C. jejuni CFU/250 ml of seeded natural waters. The other methods, plating the final filter face-up or preincubation of the filter in an enrichment medium, were not as sensitive. The technique described above could be useful in the routine monitoring of finished waters for C. jejuni or during investigations of suspected waterborne outbreaks for water of low turbidity.     

Procedure for increased recovery of Campylobacter jejuni from inoculated unpasteurized milk

Different treatments were applied to Campylobacter jejuni-inoculated unpasteurized milk to identify means of enhancing the survival of the organism in refrigerated (4 degrees C) samples. The greatest survival occurred in milk supplemented with 0.01% sodium bisulfite and held under an atmosphere of 100% nitrogen (bisulfite-nitrogen), in most instances allowing isolation of C. jejuni from highly contaminated milk 15 or more days longer than from unsupplemented milk held in air (21% oxygen). Although a larger amount of Campylobacter was consistently recovered from milk treated with bisulfite-nitrogen, similar isolation rates (qualitative) resulted from milk stored in air and supplemented with 0.01% sodium bisulfite and 0.15% sodium thioglycolate when analyzed within 12 days after sampling. Milk samples to be transported and assayed at a later date would best be held refrigerated (4 degrees C) and supplemented with 0.01% sodium bisulfite and either 0.15% sodium thioglycolate or an atmosphere of 100% nitrogen.     

Comparison of methods for isolating Campylobacter jejuni from raw milk.

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental water

Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained.     

Methods for isolating Campylobacter jejuni from low-turbidity water.

Membrane filtration methods were developed and evaluated for the quantitative recovery of Campylobacter jejuni from environmental waters of low turbidity. The best procedure studied involved passaging the test water through a filter (pore size, 0.45 micron) and plating it facedown on Campylobacter-selective agar. The filter was removed after overnight incubation, and the plate was streaked for isolation and then reincubated. This method, with or without prefiltration through 5.0- and 0.6-micron-pore-size membranes consistently resulted in the recovery of 30 C. jejuni CFU/250 ml of seeded natural waters. The other methods, plating the final filter face-up or preincubation of the filter in an enrichment medium, were not as sensitive. The technique described above could be useful in the routine monitoring of finished waters for C. jejuni or during investigations of suspected waterborne outbreaks for water of low turbidity.     

The isolation and characterization of Campylobacter jejuni subsp. jejuni from domestic geese (Anser anser)

AIMS: The objectives of this study were to determine the presence of thermophilic Campylobacter spp. in free range domestic geese, and to characterize isolated strains using phenotyping criteria and SDS-PAGE of whole-cell proteins. METHODS AND RESULTS: Forty cloacal swabs from two different flocks of domestic geese were examined. All Camp. jejuni strains isolated from geese were biotyped using the Lior biotyping scheme. Twelve Camp. jejuni isolates were also tested for their susceptibility to 17 different antibacterial agents by a disc diffusion METHOD: Fourteen of the isolates were also subjected to SDS-PAGE. All of the geese examined were found to harbour Camp. jejuni. Six geese carried more than one species of Campylobacter. All strains examined were susceptible to various antibiotics but resistant to penicillin G and cephalothin. Eleven strains (92%) were resistant to sodium cefuroxime, and eight (67%) were resistant to cloxacillin, ampicillin and colistin sulphate. Three strains (25%) were resistant to tetracycline, and one strain was resistant to sulfamethoxazole/trimethoprim and kanamycin. Nine strains were subtyped as Camp. jejuni subsp. jejuni biotype II and the remaining ones as biotype I. There were 96% and 100% similarities between all the strains examined by SDS-PAGE. CONCLUSION: This study showed that Camp. jejuni were common in the intestinal tract of domestic geese. SIGNIFICANCE AND IMPACT OF THE STUDY: Geese should be considered as potential reservoirs for human and animal campylobacteriosis. The antibiotic resistance data from this study also showed that fluoroquinolone resistance, which appears to be a problem in poultry isolates in some countries, is not yet a problem in these geese.     

Evaluation of filters for recovery of Campylobacter jejuni from water

Campylobacter jejuni has been incriminated in several large waterborne outbreaks, but it has rarely been isolated from water itself. Better methodology is needed for the isolation of C. jejuni from water. We evaluated three types of 0.45-micron microporous filters and three different pore sizes of positively charged depth filters for their ability to recover C. jejuni from seeded, sterile tap and surface water. The microporous filters tested were Millipore HA, Gelman GN6, and Zetapor. Three pore sizes of Zeta Plus depth filters (05S, 30S, and 50S) were evaluated by using an adsorption-elution technique. The overall percent recovery in both tap and surface water by microporous filters was: Zetapor, 66%; Millipore HA, 33%; and Gelman GN6, 33%. Adsorption-elution with Zeta Plus 50S allowed 89% recovery of C. jejuni. These data suggest that both the positively charged Zetapor microporous filter and the Zeta Plus 50S depth filter are effective filters for the recovery of C. jejuni from water.     

Efficacy of supplemented buffered peptone water for the isolation of Campylobacter jejuni and C. coli from broiler retail products

Broiler retail samples (n=113) were analyzed to determine (i) the effectiveness of buffered peptone water (BPW) supplemented with blood and antibiotics for the isolation of Campylobacter jejuni and C. coli, (ii) if a 1:4 enrichment ratio performs similarly as a 1:9 ratio, and (iii) if BPW is similar to Bolton broth for enumeration of Campylobacter spp. in retail broiler meat using the most probably number (MPN) procedure. Chi-square comparison showed that BPW performed similarly as Bolton broth (P< or =0.05) for Campylobacter isolation in breast tenders, boneless breasts, split breasts and skin samples. However, BPW showed a lower detection rate (P> or =0.05) for thighs and boneless thighs. When the results were combined, BPW performed similarly as Bolton broth for the isolation of Campylobacter spp. (P< or =0.05). BPW at an enrichment ratio of 1:4 was statistically similar to Bolton broth or BPW at a ratio of 1:9. No differences were observed between the MPN data from Bolton broth and the MPN data from BPW (P< or =0.50). A multiplex PCR assay revealed that ca. 48% of the isolates obtained from Bolton broth and 59% of the isolates obtained with BPW were C. coli. Both Bolton broth and BPW allowed for the growth of C. jejuni and C. coli from the same sample. Remarkably, a large genomic variability was observed by PFGE analysis of the isolates collected from the same sample with Bolton broth or BPW, which confirms that more than one genotype can successfully multiply during enrichment and be recoverable on agar plates. These findings suggest that BPW could be used as an enrichment medium for isolation of Campylobacter from retail broiler samples. The implications of the high number of C. coli isolates found in this study is discussed.