Microtitre plate hybridization system for detection of thermophilic Campylobacter rRNA

A microtitre plate nucleic acid probe hybridization system was developed for the detection of ribosomal RNA from thermophilic Campylobacter (Camp. jejuni, Camp. coli, Camp. lari and Camp. upsaliensis). A specific DNA probe obtained by amplification of 23S rRNA sequences using the polymerase chain reaction technique was immobilized on a microtitre plate, and used for hybridization with target 23S rRNA from cell lysates. The RNA-DNA hybrids thus formed in the wells were detected by an immunoenzymatic assay using a monoclonal antiRNA-DNA hybrid antibody. The sensitivity of this system was 2.7 x 10(4) cells ml(-1). This simple, sensitive and inexpensive hybridization and immunoenzymatic assay system should facilitate the detection of Campylobacter in food and clinical samples.     

Advantages of peptide nucleic acid oligonucleotides for sensitive site directed 16S rRNA fluorescence in situ hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli and Campylobacter lari

Traditionally fluorescence in situ hybridization (FISH) has been performed with labeled DNA oligonucleotide probes. Here we present for the first time a high affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting thermotolerant Campylobacter spp. using FISH. Thermotolerant Campylobacter spp, including the species Campylobacter coli, Campylobacter jejuni and Campylobacter lari, are important food and water borne pathogens. The designed PNA probe (CJE195) bound with higher affinity to a previously reported low affinity site on the 16S rRNA than the corresponding DNA probe. PNA also overcame the problem of the lack of affinity due to the location of the binding site and the variation of the target sequence within species. The PNA probe specificity was tested with several bacterial species, including other Campylobacter spp. and their close relatives. All tested C. coli, C. jejuni and C. lari strains were hybridized successfully. Aging of the Campylobacter cultures caused the formation of coccoid forms, which did not hybridize as well as bacteria in the active growth phase, indicating that the probe could be used to assess the physiological status of targeted cells. The PNA FISH methodology detected C. coli by membrane filtration method from C. coli spiked drinking water samples.     

A novel polymerase chain reaction assay for the detection and speciation of thermophilic Campylobacter spp.

A novel PCR amplification method is described which is specific for the thermophilic, enteropathogenic species Campylobacter jejuni, Camp. coli and Camp. upsaliensis. Rapid, accurate speciation of amplified strains is possible on the basis of restriction fragment length polymorphisms of PCR products digested with three restriction enzymes, Alu I, Dde I and Dra I. The sensitivity of detection is 25 cfu in water, and 2 x 10³ cfu in full cream milk. An epidemiological application of the assay in detecting non-culturable campylobacters from a contaminated potable water supply is described.     

Sensitivity of Campylobacter spp. to irradiation in poultry meat

The sensitivity of Campylobacter jejuni (three strains), Camp. coli (three strains), Camp. fetus (one strain) and Camp. lari (one strain) to irradiation in poultry meat was investigated. There was no significant difference in the counts obtained on Blood or Skirrows agar. Preston agar gave a significantly lower recovery of the pathogens after irradiation so these results were not included in calculations of D ₁₀ values. The D ₁₀ values ranged from 0.12 to 0.25 kGy and there was a significant difference in the radiation sensitivity between different Campylobacter spp. and within strains of the same species. These values indicate that Campylobacter spp. are more radiation-sensitive than Salmonella and Listeria monocytogenes irradiated under similar conditions. Therefore irradiation treatments suggested to eliminate the latter from poultry carcasses would also be sufficient to remove Campylobacter.     

Identification of thermophilic Campylobacter spp. by phenotypic and molecular methods

AIMS: The differences between phenotyping and genotyping (polymerase chain reaction- restriction fragment length polymorphism) of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis were assessed. METHODS AND RESULTS: A total of 51, 63 and 88 strains from dogs, pigs and humans, respectively, were examined. The strains were first typed by biochemical methods, then by PCR-RFLP using AluI and Tsp509I. None of the strains were typed as Camp. lari by the PCR-RFLP. The biggest differences were found in the identification of Camp. jejuni and Camp. coli. The main discrepancies were caused with the hippurate hydrolysis test and sensitivity to cephalothin and nalidixic acid. Strains which were identified biochemically as Camp. coli and by digestion with AluI as Camp. jejuni (eight strains) were tested for the presence of the hippuricase gene. CONCLUSION: The PCR typing results showed the presence of the hippuricase gene as unique to Camp. jejuni. SIGNIFICANCE AND IMPACT OF THE STUDY: A reliable identification of Campylobacter spp. should be supplemented with a molecular method.     

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.     

A PCR assay for the detection of Campylobacter jejuni and Campylobacter coli in water

A PCR assay has been developed for the detection of Campylobacter jejuni and Camp. coli in water samples. The sample is filtered through a membrane which is subjected to sonication to release the impacted cells. After removal of the filter from the cell suspension and a freeze/thaw cell lysis step, a semi-nested PCR is carried out on the filtrate using the primers CF02, CF03 and CF04 ( Camp. jejuni fla and flaB gene sequences). Incorporation of a sonication stage allows removal of the filter membrane since they have been shown to inhibit the PCR. In experiments with spiked water samples (20 ml) a theoretical sensitivity of 10–20 Campylobacter cells ml^-1 was achieved. Using a sample volume of 100 ml this sensitivity can be increased to approximately 2 Campylobacter cells ml^-1.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction.

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.     

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.     

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.     

Development of real-time PCR and hybridization methods for detection and identification of thermophilic Campylobacter spp. in pig faecal samples

AIMS: To develop a real-time (rt) PCR for species differentiation of thermophilic Campylobacter and to develop a method for assessing co-colonization of pigs by Campylobacter spp. METHODS AND RESULTS: The specificity of a developed 5' nuclease rt-PCR for species-specific identification of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and of a hipO gene nucleotide probe for detection of C. jejuni by colony-blot hybridization were determined by testing a total of 75 reference strains of Campylobacter spp. and related organisms. The rt-PCR method allowed species-specific detection of Campylobacter spp. in naturally infected pig faecal samples after an enrichment step, whereas the hybridization approach enhanced the specific isolation of C. jejuni (present in minority to C. coli) from pigs. Conclusions: The rt-PCR was specific for Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis and the colony-blot hybridization approach provided an effective tool for isolation of C. jejuni from pig faecal samples typically dominated by C. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Species differentiation between thermophilic Campylobacter is difficult by phenotypic methods and the developed rt-PCR provides an easy and fast method for such differentiation. Detection of C. jejuni by colony hybridization may increase the isolation rate of this species from pig faeces.     

Identification of campylobacteria isolated from Danish broilers by phenotypic tests and species-specific PCR assays

AIMS: To validate a phenotypic Campylobacter species identification method employed to identify campylobacters in broilers by comparison with campylobacterial species identification using various species-specific PCR analyses. METHODS AND RESULTS: From a collection of 2733 phenotypically identified campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while other species identified were Helicobacter pullorum, Camp. lari and Camp. upsaliensis. However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged to H. pullorum. It was also notable that 2-6% of the cultures were, in fact, mixed cultures. CONCLUSIONS: The phenotypic identification scheme employed failed to appropriately differentiate Campylobacter species and particularly to identify the closely related species, H. pullorum. SIGNIFICANCE AND IMPACT OF THE STUDY: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers.     

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens isolated at an Irish poultry processing plant

AIMS: The antibiotic susceptibility of Campylobacter jejuni and Campylobacter coli isolates from broiler chickens were determined in order to evaluate the level of antibiotic resistance of Campylobacter species in the Irish poultry industry. METHODS AND RESULTS: Seventy-eight Camp. jejuni and 22 Camp. coli strains were examined for susceptibility to eight antibiotics using the disc diffusion assay. The highest level of resistance of the Camp. jejuni isolates was recorded to ampicillin (35.9%), followed by 20.5% to tetracycline, 20.5% to naladixic acid, 17.9% to ciprofloxacin, 10.2% to erythromycin, 2.5% to streptomycin and 1.2% to kanamycin. Multidrug resistance to two or more antibiotics was seen for 30.7% of Camp. jejuni strains. Resistance of the Camp. coli isolates was shown to ampicillin (9%) and tetracycline (18.2%). CONCLUSIONS: The majority of Camp. jejuni strains were susceptible to antibiotics commonly used for human therapy. Camp. coli strains showed very low resistance levels and were susceptible to six of the eight antimicrobial agents studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Levels of Camp. jejuni and Camp. coli antimicrobial resistance in Irish poultry production was assessed to determine the current situation in Ireland. The prevalence of antibiotic resistance of Campylobacter strains isolated from broiler chickens was low.     

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.     

Factors affecting the pressure resistance of some Campylobacter species

AIMS: To compare pressure resistance between strains of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus, and to investigate the effect of suspending medium on pressure resistance of sensitive and more resistant strains. METHODS AND RESULTS: Six strains of C. jejuni and four each of C. coli, C. lari and C. fetus were pressure treated for 10 min at 200 and 300 MPa. Individual strains varied widely in pressure resistance but there were no significant differences between the species C. jejuni, C. coli and C. lari. Campylobacter fetus was significantly more pressure sensitive than the other three species. The pressure resistance of C. jejuni cultures reached a maximum at 16-18 h on entry into stationary phase then declined to a minimum at 75 h before increasing once more. Milk was more baroprotective than water, broth or chicken slurry but did not prevent inactivation even of a resistant strain at 400 MPa. CONCLUSIONS: Pressure resistance varies considerably between species of Campylobacter and among strains within a species, and survival after a pressure challenge will be markedly influenced by culture age and food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the strain variation in pressure resistance and protective effects of food, Campylobacter sp. do not present a particular problem for pressure processing.     

Yoghurt: an unlikely source of Campylobacter jejuni/coli

Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2–5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 10⁵–10⁸ cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3–5 logs in 30 min. Strong inorganic acid HC1, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni , it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.     

空肠弯曲菌的磁捕获_-荧光PCR检测方法的建立

为提高畜禽类食品中空肠弯曲菌的检出率和灵敏度,应用抗血清和磁性微珠首次制备弯曲菌免疫磁珠,利用弯曲菌免疫磁珠直接捕获检样中目的菌,不需要增菌培养;通过荧光PCR检测鞭毛蛋白A(flaA)基因和/或马尿酸酶(hipO)基因,首次建立空肠弯曲菌的磁捕获-荧光聚合酶链反应(IMC-FPCR)方法.IMC-FPCR法检测空肠弯曲菌方法简便易行,可在24h内完成,特异性好,检测低限达到10cfu/mL,抗干扰性强.IMC-FPCR方法可望解决非可培养状态的空肠弯曲菌检测难题,是一种适用于检验检疫、卫生防疫和农产品安全检验等领域的快速方法.     

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.     

Enumeration of Campylobacter in New Zealand recreational and drinking waters

AIMS: To use a published polymerase chain reaction (PCR) method for the detection and identification of thermotolerant Campylobacter species (Camp. jejuni, Camp. coli and Camp. lari) in tandem with a Most Probable Number (MPN) technique to enumerate these species in water samples. METHODS AND RESULTS: An initial study of 42 river water samples compared the use of conventional culture and PCR methods for the detection of Campylobacter in MPN enrichment tubes. It was found that all samples positive by culture were also positive by PCR. Thirty-seven percent more MPN tubes were positive by PCR compared with culture. The MPN/PCR technique was subsequently applied to 96 additional samples collected from rivers, drinking, roof and shallow ground water. Campylobacter was especially prevalent in river water (60% positive) and shallow ground water (75% positive) samples. Drinking water (29.2% positive) and roof water (37.5% positive) also contained Campylobacter, but the numbers detected were very low (maximum 0.3 and 0.56 MPN 100 ml-1, respectively). CONCLUSION: River waters contained Campylobacter at higher levels than any other water type and in a high percentage of the samples. Although Campylobacter was present in treated drinking water, the levels detected were low. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that water may act as a significant transmission route for human campylobacteriosis.