A blood-free enrichment medium for growing Campylobacter spp. under aerobic conditions

Detection limits for Campylobacter jejuni strains JH93 and ATCC 29428 in a new blood-free enrichment broth (BFEB) were investigated under aerobic conditions. Cultures of Camp. jejuni were inoculated into 50 ml BFEB containing 10% food homogenate in 50 ml screw-cap tubes. After 24 h enrichment under aerobic conditions, Camp. jejuni were isolated on four selective agar media. The least squares means of the detection limit 50% endpoint (DL₅₀) values were 0·4 (plain BFEB), 0·9 (crabmeat), 1·7 (mushroom), 1·7 (raw milk) and 2·1 (oyster) colony forming units (cfu) 5 g⁻¹ food. The efficiency of the BFEB was significantly affected ( P < 0·05) by food type and bacterial strain. Overall, the BFEB enrichment compared favourably with the existing US Food and Drug Administration method under modified atmosphere. In addition, the BFEB method did not require the use of blood, special equipment or Oxyrase^® to reduce oxygen tensions.     

Eight enrichment broths for the isolation of Campylobacter jejuni from inoculated suspensions and ground pork

Aims:  The efficiency of eight enrichment broths for the selective isolation of Campylobacter jejuni was compared to identify an optimal enrichment broth.
Methods and Results:  Brucella-FBP, Preston, Doyle and Roman, modified CCD (mCCD), Park and Sanders, Bolton, Hunt and Radle and Hunt broths were compared for their recovery of (i) Camp. jejuni in suspension, (ii) Camp. jejuni from inoculated ground pork, (iii) heat-injured Camp. jejuni (55°C for 20 min) in suspension and (iv) heat-injured Camp. jejuni from inoculated ground pork. Hunt broth and Bolton broth showed the highest and most rapid enrichment efficacy for the cell suspensions and ground pork, respectively. Preston, Park and Sanders and mCCD broths had relatively high enrichment efficiencies, while Brucella-FBP broth was significantly inferior to the other broths ( P  < 0·05).
Conclusions:  Cell recovery from the eight enrichment broths was dependent on the sample type and the state of the cells. The use of the appropriate broth is important for the rapid and efficacious enrichment of Camp. jejuni . In particular, heat-injured Camp. jejuni require a longer cultivation time and a suitable enrichment broth.
Significance and Impact of the Study:  The results from the present study provide information for selecting the most appropriate enrichment broth for Camp. jejuni and may contribute to improved detection methods for the organism.     

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.     

A MODIFIED ANAEROGENTM SYSTEM FOR GROWTH OF CAMPYLOBACTER SPP

A simple, rapid procedure to generate an oxygen-reduced atmosphere suitable for growing Campylobacter jejuni was investigated. A modified AnaeroGen^TM system (MAS), consisting of a single AnaeroGen^TM anaerobic sachet AM35 (Oxoid Unipath Ltd., Basingstoke, UK) activated in a 9 L anaerobic jar (BBL, Cockeysville, MD), was evaluated and compared with the conventional gassed jar system described in the 8th edition of the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). Enriched cultures of C. jejuni (six replicates at each of five inoculum levels: 10^-2 to 10² cfu/mL in artificially contaminated raw milk, raw oyster, crab meat, or mushroom were streaked in duplicate onto four different selective isolation agars for simultaneous incubation in MAS and in the BAM system. No significant differences (p > 0. 05) in recovery rates of C. jejuni were observed for the two systems. The type of isolation agar used did not affect these recovery rates. The quality of growth of C. jejuni at 24, 48 or 72 h was similar in both systems. MAS successfully reduced atmospheric oxygen to a level suitable for the growth of C. jejuni. It was simple and rapid compared to the BAM system, and cost-effective compared to the Oxoid CampyGen^TM system specifically designed for the growth of Campylobacter spp.     

Comparison of Four Enrichment Media in the Recovery of Campyloracter Jejuni

The effectiveness of 4 enrichment media for the recovery of low levels of inoculated cells of Campylobacter jejuni was evaluated. The media contained antibiotics or antibiotics and bile acids as selective compounds. Three of the media recovered most of the inoculated low numbers of 6 C. jejuni strains. In the 3 media the growth rate of 3 strains, indicated by the increase in the log number of cells during 24 h or 48 h incubation at 42 ° C, was about the same as in the control medium without selective compounds. The same 3 media also recovered a low number of Campylobacter cells from artificially contaminated raw milk or ground meat samples. The enrichment medium B containing 40 I.U. Colistin, 5 μg novobiocin, 2 mg Na-cholic acid and 50 mg cycloheximide per ml was inhibitory for most Campylobacter strains studied.     

Effect of temperature, duration of incubation, and pH of enrichment culture on the recovery of Campylobacter jejuni from eviscerated market chickens

To establish an enrichment system of high efficiency for recovery of Campylobacter jejuni from market chickens, the effects of the temperature, duration of incubation, and pH of the enrichment culture on the isolation of the bacterium were evaluated. Whole chickens or chicken parts in plastic bags were individually rinsed, and the washings filtered through cheesecloth. The cells were separated from the washings by centrifugation, and the pellet was inoculated into 100 mL of enrichment broth. Isolation of C. jejuni from poultry samples was significantly increased by incubating these samples in an enrichment medium at 42 degrees C as opposed to 35 degrees C; for 48 h as opposed to 24 h or 72 h; and at pH 7.0 as opposed to pH 6.0, 6.5, 7.5, or 8.0.     

A METHOD FOR ISOLATION OF CAMPYLOBACTER SPP. (JEJUNI, COLI AND LARI) FROM SHELLFISH AND THE MARINE ENVIRONMENT

A method has been developed to detect thermophilic species of Campylobacter in shellfish, marine and tributary waters, sediment and farm runoff by-products such as manure and silage. The method consists of a 48 h enrichment incubation and subcultured to selective agars. Presumptive colonies confirmed with a latex agglutination (antibodies) to common flagellar antigens of C. jejuni, C. coli and C. lardi. Over an 8 year period, West Coast estuaries (Washington, Oregon, and California) were sampled, resulting in analysis of a total of 512 samples. Results suggest that Campylobacter spp. are well distributed in the marine environment. Two enrichment broths were compared for the recovery of campylobacters from environmental samples. The method described in the Food and Drug Administration Bacteriological Analytical Manual (FDA/BAM) (1984), was compared to a modified method. Use of the modified method described here resulted in higher recovery rates of Campylobacter spp. Recoveries of campylobacters from sediment, shellfish, and water were 10,13, and 28% higher for the modified method, respectively.     

选择性增菌液对单核增生性李斯特氏菌检出效果的比较

为了了解食品中单核增生李斯特氏茵(Listeria monocytogenes)的污染状况,比较不同选择性增茵方法对单核增生李斯特氏茵的检出效果,并进一步比较不同增茵方法在不同类食品中检出单核增生李斯特氏茵的差异性,进而确定特定食品最合适的增茵方法,随机采集本市生鲜肉、水产品、果蔬及冷冻食品4类135份食品.采用国标LB二次增茵法、EB法、最新改良FDA法及Fraser肉汤增菌法进行增菌,采用PALCAM选择性平板进行分离,先用行标多重PCR法进行初步验证后再进行国标生化鉴定.4种方法共检出单核增生李斯特氏茵23株,其中LB二次增菌法检出5株、Fraser肉汤增菌法检出6株、EB法检出5株、最新改良FDA法检出7株.结论是4种方法总的检出率没有较大的差异性,但对于不同类食品的检出率有所不同.     

Detection of Campylobacter jejuni and Camp. coli in chicken faecal samples by PCR

H.N. RASMUSSEN, J.E. OLSEN, K. JØRGENSEN AND O.F. RASMUSSEN. 1996. PCR primers were selected from the flagellin gene sequences flaA and flaB of Campylobacter coli to amplify DNA from Camp. jejuni and Camp. coli. When the PCR products were analysed by hybridization to an internal probe immobilized in microtitre wells, positive reactions were observed only for strains of Camp. jejuni and Camp. coli. The assay was used to analyse 31 chicken faecal samples. Full correspondence was found between the PCR assay conducted on the enriched cultures and the standard culture method. When analysing the transport medium prior to enrichment, the PCR assay detected nine of 11 culture positive samples.     

The prevalence of campylobacters and arcobacters in broiler chickens

Chicken carcasses from a supermarket and from a poultry abattoir were examined using methods designed to isolate as many strains of campylobacters and related organisms as possible. Strains of arcobacter, but no campylobacters, were isolated from every carcass after enrichment. Campylobacter jejuni subsp. jejuni was isolated from all carcasses examined by direct plating and other Campylobacter -like strains were isolated from nine out of 15 abattoir carcasses by direct plating but not after enrichment. Only the Camp. jejuni subsp. jejuni strains could be identified to species level using a readily available identification scheme and/or a commercial identification kit. Examination of caecal contents from the 15 abattoir poultry yielded Camp. jejuni subsp. jejuni and Campylobacter -like strains from 15 and eight by direct plating, and from six and nine after enrichment, respectively. Four sites in the intestine of the abattoir birds (60 samples) were examined for arcobacters and only one strain was isolated. This indicates that arcobacters are probably not normal inhabitants of the poultry intestine. Poultry is a rich source of other campylobacteria besides the thermophilic Campylobacter spp.     

Selected enrichment broths for recovery of Campylobacter jejuni from foods.

We attempted to shorten the required time for enrichment broth culture for the isolation of Campylobacter jejuni. Enrichment broths described by Doyle and Roman and Park and Stankiewicz and one developed during this study were compared for ability to isolate C. jejuni from raw chicken carcasses. Our medium was a modification of that of Doyle and Roman with the addition of filter-sterilized FBP (0.2% ferrous sulfate, 0.025% sodium metabisulfite, 0.05% sodium pyruvate), 0.1% sodium lauryl sulfate, and 0.075% agar. Initially, laboratory strains were employed in the development of this medium. Subsequently, an indigenous load of C. jejuni obtained from chickens was used to compare media. Isolation rate comparisons were as follows: direct plating, 40%; Doyle and Roman broth, 45% at 7 h and 61% at 16 h; Park and Stankiewicz broth, 53% at 7 h and 60% at 16 h; our broth, 48% at 7 h and 50% at 16 h. In addition to having the highest isolation rate, the enrichment broth of Doyle and Roman showed greatest selectivity. Our inoculation method of indigenous bacteria provided a controlled means for comparison of isolation procedures.     

Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method.

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

Four of five strains of Campylobacter jejuni survived in chicken meat stored at -18 degrees C for 12 months. Direct plating of samples was superior to the most probable number technique for enumerating C. jejuni. Enrichment culture using the Doyle & Roman enrichment method resulted in the highest rates of detection. Packaging under an atmosphere of CO2 did not substantially influence survival.     

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

Four of five strains of Campylobacter jejuni survived in chicken meat stored at — 18C for 12 months. Direct plating of samples was superior to the most probable number technique for enumerating C. jejuni. Enrichment culture using the Doyle & Roman enrichment method resulted in the highest rates of detection. Packaging under an atmosphere of CO₂ did not substantially influence survival.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle.

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

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.     

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.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

Evaluation of the effect of temperature and nutrients on the survival of Campylobacter spp. in water microcosms

Batch microcosms containing various water types (de-ionized and river water with or without sediment), incubated at a range of temperatures (5-37 degrees C), were used to facilitate a comparative evaluation of the significance of such variables and their interactions upon the collective and individual survival of four species of thermophilic Campylobacter. All variables significantly influenced (P < = 0.031) population decay rates. Minimal decay for the group was identified at low temperatures (5 degrees C) in river water, i.e. nutrient-containing microcosms. Collective decay rates within river water microcosms were significantly decreased (P = 0.03) from those observed in de-ionized water, particularly at environmental temperatures (5 and 15 degrees C). However, the increased nutrient levels observed in sediment-containing microcosms did not significantly (P = 0.41) reduce population decay rates. Overall, Camp. jejuni populations demonstrated the most resilience to the environmental stressors evaluated, with the exception of 15 degrees C where Camp. lari was the most persistent. Campylobacter coli and Camp. upsaliensis demonstrated comparable survival characteristics but were less resilient than Camp. jejuni and Camp. lari. These observations identify the suitability of water systems as a reservoir and medium for Campylobacter infection, and potentially identifies Camp. jejuni and Camp. lari as the main protagonists of water-mediated campylobacteriosis.