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.     

Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Application of conductance techniques to the detection of thermophilic Campylobacter spp. in river water

A study of basal media identified Campylobacter enrichment broth, with (CEB+) and without (CEB) antibiotic supplement, as a suitable medium for the detection and enumeration of Campylobacter jejuni, C. coli and C. lari within aqueous samples via conductance methodology. Despite apparent differences in conductivity profiles between species in the presence of antibiotics, no significant differences (P<0.05) were detected between detection times for each species tested. CEB+ was successfully employed within a combined enrichment and conductance protocol to the detection of C. jejuni from river water at a concentration of 1 CFU ml⁻¹ from 83% of samples in under 39 h and thus demonstrated an improvement over an applied conventional membrane filtration technique.     

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.     

Urease-positive thermophilic strains of Campylobacter isolated from seagulls (Larus spp.)

Three strains of urease-positive thermophilic Campylobacter (UPTC), designated A1, A2 and A3, were identified by biochemical characterization after isolation from faeces of seagulls in Northern Ireland in 1996. The biochemical characteristics of the strains were identical to those of strains described previously. Analysis by pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI and SmaI demonstrated that the respective PFGE profiles were indistinguishable. The PFGE analysis also suggested that the genomes were approximately 1810 kb in length. This is the first example of the isolation of UPTC from flying homoiothermal animals, i.e. from seagulls (Larus spp.).     

Differentiation between thermophilic Campylobacter species by species-specific antibodies.

The four species of thermophilic campylobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari, are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.     

Identification and characterization of thermophilic Synechococcus spp. isolates from Asian geothermal springs

Two thermophilic cyanobacterial strains, Ts and Bs, collected from Asian geothermal springs were identified morphologically and phylogenetically as Synechococcus in the order Chroococcales and were isolated into axenic cultures. In addition to the high similarities between their full 16S rRNA gene sequences, both strains also shared similar pigment profiles and fatty acid compositions but with varied ratios. Strain Ts had elevated levels of photoprotective pigments such as carotenoid and scytonemin even after prolonged culture under identical laboratory conditions, whereas strain Bs produced more chlorophyll a per unit cell volume, perhaps resulting from UV adaptation in the natural habitats. In addition, strain Ts had more content than strain Bs in terms of the total fatty acids and the proportion of unsaturated fatty acids. Neither isolate was able to fix nitrogen, and they had zero susceptibility to ampicillin and streptomycin.     

Colonization of chicks by non-culturable Campylobacter spp.

Six suspensions of non-culturable Campylobacter spp. were administered by gavage to day-of-hatch chicks. Four non-culturable isolates of Campylobacter spp. were found to colonize low numbers (5/79) of 1-week-old chicks, while two isolates did not (0/30). The original and recovered Campylobacter spp. isolates were serotyped and examined by restriction enzyme analysis. Evidence of clonality of two Camp, jejuni isolates was demonstrated.     

Culturability, injury and morphological dynamics of thermophilic Campylobacter spp. within a laboratory-based aquatic model system

AIMS: To study the survival processes of thermophilic Campylobacter spp. within a modelled aquatic system and particularly the involvement and survival potential of viable but non-culturable forms. METHODS AND RESULTS: The survival and morphological characteristics of populations of thermophilic Campylobacter species exposed to simulated aquatic conditions were examined using a combination of cultural and microscopic techniques. Populations underwent progressive decay when exposed to simulated aquatic conditions. The rates of population decay were observed to be significantly greater at the higher temperature (20 degrees C) with a rapid transition of the dominant sub-populations from non-stressed to dead cells occurred within 3 days. At 10 degrees C the rate of culturability loss was much reduced with substantial development (approx. 80% of total population) of viable but non-culturable (VBNC) populations by all species within 3 days, declining to represent approximately 5-25% of the total population at day 60. Significant differences (P < 0.001) were identified between decay rates as a consequence of different species, sub-populations and temperature but not between sub-populations of different species. Morphological variants including spiral, elongated spirals and rods, short rods and coccoid forms were identified. The endpoints of morphological transition were temperature-independent and isolate-specific yet the rate of morphological transition was directly related to temperature and approximately equivalent between species. CONCLUSION: The VBNC state is a transitory stage in the degeneration of Campylobacter population within the aquatic environments simulated during this study. SIGNIFICANCE AND IMPACT OF THE STUDY: VBNC cells form the most persistent, viable, potentially pathogenic sub-population of Campylobacter populations exposed to aquatic stress conditions.     

Antimicrobial resistance and genomic screening of clinical isolates of thermophilic Campylobacter spp. from south-east Queensland,Australia

AIM: To screen 90 clinical isolates of thermophilic Campylobacter species for putative resistance to ampicillin, erythromycin and tetracycline and perform numerical analysis to determine isolate relatedness. METHODS AND RESULTS: Disc diffusion, E-test MIC and agar dilution methods were performed. Disc diffusion testing showed 87 (97%) isolates appeared resistant to ampicillin at 10 microg; 14 (16%) resistant to tetracycline at 30 microg; and three (3.4%) resistant to erythromycin at 15 microg. E-test MICs showed a range of 0.5 to >256 mg l(-1) for ampicillin; 16 to >256 mg l(-1) for tetracycline; and >256 mg l(-1) for erythromycin. E-test showed 68% correlation (+/-1 log2 dilution) with agar dilution for ampicillin, 100% for erythromycin and 64% for tetracycline. Disc diffusion testing showed 100% correlation with agar dilution for erythromycin and tetracycline, and 77% for ampicillin. Numerical analyses of restriction endonuclease (RE) fragment profiles suggested a high level of isolate variation. CONCLUSION: The incidence of resistance of thermophilic Campylobacter spp. to erythromycin and tetracycline is low in south-east Queensland. SIGNIFICANCE AND IMPACT OF THE STUDY: Disc diffusion susceptibility testing may be used to screen thermophilic Campylobacter spp. for putative resistance to erythromycin and tetracycline. Agar dilution should be used to determine ampicillin susceptibility.     

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.     

Characterization of thermophilic Campylobacter. II. Enzymatic profiles

The enzymatic profiles of 234 wild strains of thermophilic Campylobacters, seven type strains ofCampylobacter species, and 18 reference strains ofCampylobacter species and Campylobacter-like organisms were studied by use of API strips. These strips allow the detection of 56 arylamidases, one transpeptidase, and ten esterases.Forty enzymes were present at least once. The mean number of enzymes per strain was 13. The enzymatic activity was usually weak. Three enzymes were present in all the strains: esterases of butyric and valeric acids, andl-phenylalanine-l-proline arylamidase. A combination of three enzymes provided a good predictive value for the species differentiation ofC. jejuni andC. coli. There were no differences in relation to the geographical origin of the strain nor to the animal species from which it was isolated. The -glutamyl transpeptidase could be used for the biotyping of the strains.A portion of this work was presented at the Second Workshop of Campylobacter Infections, held in Brussels, Belgium, in September 1983.     

Typings of Campylobacter jejuni isolated from patients of 2 outbreak cases by genotypic and phenotypic methods

We compared Campylobacter jejuni strains isolated from the patient stools associated with two food-borne diarrheal outbreak cases by the serotypic methods (Lior and Penner systems) and the genotypic methods (restriction fragment length polymorphism (RFLP) of flaA gene and pulsed-field gel electrophoresis (PFGE)). Fla-RFLP was based on the digestion of 410 bp DNA fragment by MboI restriction enzyme amplified from a 5' portion of C. jejuni flaA gene. Six distinctive fla-RFLP patterns were identified by examining 29 serotype reference strains and 58 strains isolated from the patients infected with C. jejuni independently. In the first outbreak case, 4 isolates were shown to be the same patterns each other by the fla-RFLP and PFGE, and by the Lior serotyping, except the Penner system that serotyped into 2 distinct types. On the other hand, in the second case, out of 10 isolates, 5 isolates were identical by the both genotypic and the both serotypic methods, and 4 isolates were not differentiated by the fla-RFLP and Penner system, but were separated into 4 types by PFGE in a little difference. The rest isolate was completely different from the other isolates by the all of methods used now. The findings suggest that the second case occurred by the infection of at least 3 different strains of C. jejuni.     

Differentiation between thermophilic Campylobacter species by species-specific antibodies

P.L. GRIFFITHS. G.S. MORENO AND R.W. A. PARK. 1992. The four species of thermophilic camplyobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari , are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.     

A survey of fluoroquinolone resistance in Escherichia coli and thermophilic Campylobacter spp. on poultry and pig farms in Great Britain

Aims: To estimate the proportions of farms on which broilers, turkeys and pigs were shedding fluoroquinolone (FQ)-resistant Escherichia coli or Campylobacter spp. near to slaughter. Methods and Results: Freshly voided faeces were collected on 89 poultry and 108 pig farms and cultured with media containing 1·0 mg l⁻¹ ciprofloxacin. Studies demonstrated the specificity of this sensitive method, and both poultry and pig sampling yielded FQ-resistant E. coli on 60% of farms. FQ-resistant Campylobacter spp. were found on around 22% of poultry and 75% of pig farms. The majority of resistant isolates of Campylobacter (89%) and E. coli (96%) tested had minimum inhibitory concentrations for ciprofloxacin of ≥8 mg l⁻¹. The proportion of resistant E. coli and Campylobacter organisms within samples varied widely. Conclusions: FQ resistance is commonly present among two enteric bacterial genera prevalent on pig and poultry farms, although the low proportion of resistant organisms in many cases requires a sensitive detection technique. Significance and Impact of the Study: FQ-resistant bacteria with zoonotic potential appear to be present on a high proportion of UK pig and poultry farms. The risk this poses to consumers relative to other causes of FQ-resistant human infections remains to be clarified.