Campylobacter helveticus sp. nov., a new thermophilic species from domestic animals: characterization, and cloning of a species-specific DNA probe.

An atypical group of thermophilic catalase-negative Campylobacter strains, the 'CH' (Swiss) group, can be recovered from faeces of domestic cats and dogs after selection by filtration, or with the antibiotic cefoperazone. This group of strains shows no relative DNA homology with any species in rRNA superfamily VI (Vandamme et al., 1991, International Journal of Systematic Bacteriology 41, 88-103) except with four thermophilic Campylobacter species, notably C. upsaliensis. The group is homogeneous and possesses a DNA base composition, cellular morphology at the electron microscope level and phenotypic properties characteristic of Campylobacter. Nonetheless it is distinct from known species of Campylobacter in terms of conventional bacteriological tests, total cellular protein profile, rRNA gene profile, and genomic DNA homology. On the basis of an integrated study of phenotype and genotype, we conclude that these bacteria constitute a previously undescribed species for which we propose the name Campylobacter helveticus sp. nov. A species-specific recombinant DNA probe was cloned from the designated type strain (NCTC 12470) for use in identification and further analysis of the epidemiology, pathogenicity and transmission of C. helveticus.     

Identification of taxonomic and epidemiological relationships among Campylobacter species by numerical analysis of AFLP profiles

Amplified fragment length polymorphism (AFLP)-based profiling was performed on 138 strains representing all named Campylobacter species and subspecies. Profiles of 15/16 species comprised 6 to greater than 100 fragments and were subjected to numerical analysis. The mean similarity of 48 duplicate, outbreak and/or 'identical' strain profiles exceeded 94%. Species were clearly distinguished at the 17.90% similarity (S-) level in the dendrogram. Subspecies of Campylobacter jejuni and Campylobacter hyointestinalis, and biovars of Campylobacter lari and Campylobacter sputorum were distinguished at higher S-levels. All outbreak or 'genetically identical' strains of C. jejuni subsp. jejuni, Campylobacter coli, C. hyointestinalis and C. sputorum clustered at S-levels >92% and were distinguished from unrelated strains. Numerical analysis of AFLP profiles is useful for concurrent identification of taxonomic and epidemiological relationships among most Campylobacter species.     

Revision of Campylobacter, Helicobacter, and Wolinella taxonomy: emendation of generic descriptions and proposal of Arcobacter gen. nov.

Hybridization experiments were carried out between DNAs from more than 70 strains of Campylobacter spp. and related taxa and either 3H-labeled 23S rRNAs from reference strains belonging to Campylobacter fetus, Campylobacter concisus, Campylobacter sputorum, Campylobacter coli, and Campylobacter nitrofigilis, an unnamed Campylobacter sp. strain, and a Wolinella succinogenes strain or 3H- or 14C-labeled 23S rRNAs from 13 gram-negative reference strains. An immunotyping analysis of 130 antigens versus 34 antisera of campylobacters and related taxa was also performed. We found that all of the named campylobacters and related taxa belong to the same phylogenetic group, which we name rRNA superfamily VI and which is far removed from the gram-negative bacteria allocated to the five rRNA superfamilies sensu De Ley. There is a high degree of heterogeneity within this rRNA superfamily. Organisms belonging to rRNA superfamily VI should be reclassified in several genera. We propose that the emended genus Campylobacter should be limited to Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and "Campylobacter upsaliensis." Wolinella curva and Wolinella recta are transferred to the genus Campylobacter as Campylobacter curvus comb. nov. and Campylobacter rectus comb. nov., respectively. Bacteroides gracilis and Bacteroides ureolyticus are generically misnamed and are closely related to the genus Campylobacter. Campylobacter nitrofigilis, Campylobacter cryaerophila, and an unnamed Campylobacter sp. strain constitute a new genus, for which the name Arcobacter is proposed; this genus contains two species, Arcobacter nitrofigilis comb. nov. (type species) and Arcobacter cryaerophilus comb. nov. Wolinella succinogenes so far is the only species of the genus Wolinella. The genus Helicobacter is also emended; Campylobacter cinaedi and Campylobacter fennelliae are included in this genus as Helicobacter cinaedi comb. nov. and Helicobacter fennelliae comb. nov., respectively. The genus "Flexispira," with "Flexispira rappini" as the only species, is closely related to the genus Helicobacter. The free-living, sulfur-reducing campylobacters do not belong to any of these genera; they probably constitute a distinct genus within rRNA superfamily VI.     

DNA base compositions and base sequence relatedness of atypical Campylobacter jejuni strains from clinical material

Abstract The relationships of nitrate-negative campylobacters (NNC) resembling Campylobacter jejuni were investigated by DNA base composition estimation (T _m method) and DNA-DNA hybridization (S1 endonuclease assay). The 8 NNC strains which were from clinical material formed a homogeneous DNA group with a high level of relatedness (approx. 70%) to typical (nitrate-positive) C. jejuni , but were less similar (approx. 35%) to Campylobacter coli , and only slightly related (≥ 10%) to Campylobacter fetus, Campylobacter laridis and Campylobacter sputorum . The NNC strains showed small but consistent genome differences from typical C. jejuni . As these differences can be correlated with several bacteriological test differences, we conclude that the NNC strains constitute a distinct subspecies within C. jejuni .     

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within +/-5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.     

The effect of different dissolved oxygen tensions on growth and enzyme activities of Campylobacter sputorum subspecies bubulus

Campylobacter sputorum subspecies bubulus was grown in batch cultures in which the dissolved oxygen tension (d.o.t) was maintained at various constant levels. At a range of d.o.t. from 0.002 to 0.05 atm, which allowed good growth (mean generation time approximately 1.5 h), L-lactate was preferentially consumed before D-lactate. L-lactate oxidation was accompanied by equimolar acetate production during exponential growth. A value for YL-lactate (g dry weight bacteria per mol L-lactate) of 54 was determined. Net acetate production stopped when C. sputorum started to use D-lactate after consumption of L-lactate. When a culture growing exponentially at the expense of L-lactate was shifted from a d.o.t. of 0.02 atm to a d.o.t. of 0.15 atm, growth was impaired, and L-lactate consumption and corresponding acetate production diminished. This decrease correlated with a loss of lactate dehydrogenase activity after the shift. Campylobacter sputorum appeared to possess cytochromes of the b- and c-type and a carbon monoxide-binding pigment. Evidence is given that the principal site of oxygen damage is lactate dehydrogenase rather than the cytochrome chain.     

Molecular cloning and site-specific mutagenesis of a gene involved in arylsulfatase production in Campylobacter jejuni.

The arylsulfatase gene from Campylobacter jejuni 81-176 encodes a predicted protein of 69,293 Da which shows no sequence similarity with other known arylsulfatases. The gene hybridizes to other Ast+ strains of C. jejuni and Campylobacter sputorum subsp. bubulus, as well as to many Ast- strains of C. jejuni.     

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.     

Routine identification of Campylobacter jejuni and Campylobacter coli from human stool samples

Correct identification of Campylobacter jejuni and Campylobacter coli isolates to the species or subspecies level is a cumbersome but nevertheless important task for a routine diagnostic laboratory. The widely used biochemical tests might be often misleading while more sophisticated phenotypic or genotypic methods are not generally available. This investigation was performed to assess the performance of common biochemical identification in comparison with species-specific PCR and gas liquid chromatography of whole cell fatty acid extracts (GLC). A total of 150 consecutive isolates from human stool samples were investigated (134 C. jejuni ssp. jejuni, 14 C. coli, two Helicobacter pullorum). From these 144, 145 and 149 isolates were correctly identified by biochemistry, GLC and PCR, respectively. Biochemical identification of all C. jejuni isolates was confirmed by PCR. GLC detected both H. pullorum strains but misidentified two C. coli strains as C. jejuni and one C. jejuni strain as C. coli. No single method can be defined as 'gold standard' for identification of C. jejuni and C. coli but a combination of techniques is needed. Therefore a stepwise identification scheme starting with biochemical reactions is suggested. All results other than C. jejuni should be confirmed by further methods. For indoxyl acetate-positive isolates species-specific PCR is recommended while GLC seems to be advantageous in indoxyl acetate-negative isolates.     

Small-scale DNA preparation for rapid genetic identification of Campylobacter species without radioisotope

A simplified and rapid genetic identification method for Campylobacter species without radioisotope was established. Three different amounts of DNA (200, 50, and 12.5 ng) extracted from each type strain of Campylobacter species with standard Marmur's procedure were spotted on a nitrocellulose filter. DNA obtained from one ml bacterial suspension at a concentration of McFarland standard turbidity No. 1 of Campylobacter fetus, C. jejuni, C. coli, and C. pylori isolates were sufficiently labeled with photo-biotin within 15 min and clearly hybridized with the type strain of the corresponding species within four to six hours. Hybridized spots were visualized with alkaline-phosphatase-conjugated streptavidin color-detection method. The reaction was usually stopped within 30 min. Atypical clinical isolates such as a nitrate-negative C. jejuni, two nalidixic acid-resistant C. jejuni, and two strains of C. fetus able to grow at 42 C, which were tentatively identified as such, were definitely identified by the simplified DNA hybridization method presented here. This method will be applicable routinely for the definite identification of atypical strains of Campylobacter species and other gram-negative bacteria difficult to identify biochemically.     

Campylobacter anatolicus sp. nov., a novel member of the genus Campylobacter isolated from feces of Anatolian Ground Squirrel (Spermophilus xanthoprymnus) in Turkey

Seventy-four Gram-negative, motile, slightly curved rod-shaped, microaerophilic, oxidase-positive and catalase-negative isolates, recovered from fecal samples of the Anatolian ground squirrel (Spermophilus xanthoprymnus) in Kayseri, Turkey, were subjected to a polyphasic taxonomic study. Results of a genus-specific PCR indicated that all isolates belonged to the genus Campylobacter. 16S rRNA gene sequence analyses revealed the closest match as Campylobacter curvus DSM 6644^T with identity levels of 96.41–96.70%. Based on the 16S rRNA gene phylogeny of the 74 isolates, six isolates (faydin-G24, faydin-G52, faydin-G105, faydin-G114, faydin-G129 and faydin-G140^T) were chosen as representatives for further characterization. The overall genome relatedness indices for the strain faydin-G140^T, compared to the most closely related type strain C. curvus ATCC 35224^T, were calculated as 15.2%, 72.5%, and 83.7% for digital DNA-DNA hybridization (dDDH), and average nucleotide identity (ANIb and ANIm), respectively. The G+C content and genome size of the strains ranged between 35.2–35.4 mol% and 1.7–1.8 Mb, respectively. Based on data obtained from the polyphasic taxonomy approach, including phenotypic characterization as well as genomic and chemotaxonomic analyses, these strains are concluded to represent a novel species, for which the name Campylobacter anatolicus sp. nov. is proposed with faydin-G140^T as the type strain (=DSM 112311^T = LMG 32238^T).     

High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting.

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

The detection of genetic variation in Leptospira interrogans serogroup ICTEROHAMORRHAGIAE by ribosomal RNA gene restriction fragment patterns

Twenty-eight isolates of catalase-negative/weak (CNW) thermophilic campylobacters from human blood and faecal cultures were characterized by one-dimensional (1-D) high-resolution SDS-PAGE of cellular proteins. A further 11 Campylobacter strains were included for reference purposes. Partial protein patterns were used as the basis for numerical analysis, which showed that all of the hippurate-positive strains had a high similarity to C. jejuni. Two subclusters were formed within C. jejuni corresponding to C. jejuni subsp. doylei (15 strains) and C. jejuni subsp. jejuni (4 strains). Most of the paediatric strains from South Africa were members of C. jejuni subsp. doylei. Hippurate-negative CNW thermophilic strains were identified as "C. upsaliensis". The analysis demonstrated that the catalase-negative C. jejuni strains were quite distinct from "C. upsaliensis" and that electrophoretic protein patterns provide an excellent criterion for the identification of subspecies within C. jejuni.     

Genotyping of clinical and chicken isolates of Campylobacter jejuni and Campylobacter coli

Genomic DNA from 58 strains of Campylobacter made up of 48 Campylobacter jejuni and ten Campylobacter coli were digested with Sma I and analysed by pulsed-field gel electrophoresis (PFGE). The cleavage of DNA by Sma I gave 22 distinct hybridization patterns. The two Campylobacter species were subtyped by PFGE. The average genomic size for C. jejuni by Sma I digestion was 1.73 Mb, while that of C. coli gave 1.7 Mb. Results from this study indicate that PFGE analysis by Sma I digested genomic DNA provides a reliable means of differentiating between and within species of Campylobacter and provides a practical approach to epidemiological studies of Campylobacter.     

Restriction endonuclease analysis, DNA relatedness and phenotypic characterization of Campylobacter jejuni and Camp. coli isolates involved in food-borne disease

Seven cases of Campylobacter infection, each of them involving two isolates, were analysed. Study of their biochemical profiles and susceptibility patterns allowed the identification of Camp. jejuni and Camp. coli isolates and the effective typing of Camp. jejuni strains into biotypes. Genotyping was carried out by comparing chromosomal DNA restriction patterns obtained by cleavage with Bgl II and Eco RV and by Southern hybridization experiments. These studies revealed clonal homogeneity between both isolates in five of the seven cases studied, indicating that in these cases Campylobacter infection was caused by a single strain. Infection with two different strains was characterized in only two of the seven cases studied, two different species belonging to Camp. coli and Camp. jejuni ssp. jejuni biotype 1 being identified. Genetic analysis proved to be the most reliable technique to achieve precise identification of strains and to elucidate clonal heterogeneity among Campylobacter isolates obtained from a single patient.     

DNA-DNA hybridization and analysis of restriction endonuclease and rRNA gene patterns of atypical (catalase-weak/negative) Campylobacter jejuni from paediatric blood and faecal cultures

Sixteen strains of atypical (catalase-weak or negative), hippurate-hydrolysing campylobacters from paediatric blood and faecal cultures were identified by DNA–DNA slot hybridization. All were closely related ( 67%) to Campylobacter jejuni and representative strains had G + C contents of 30 ± 1 mol%. Numerical analysis of chromosomal DNA Hae III digest patterns revealed two clusters of strains at the 55%S level corresponding to C. jejuni subsp. jejuni and C. jejuni subsp. doylei ; most strains belonged to the latter subspecies. No two strains had identical patterns but within each subspecies two subgroups were identifiable, corresponding to Lior biotypes I and II. Southern blot hybridization analysis with a 16 + 23S rRNA cistron probe from Escherichia coli also showed differences between the various strains, and in a numerical analysis three groupings were formed at 70%S corresponding to C. jejuni subsp. jejuni Lior biotypes I and II, and C. jejuni subsp. doylei . Four of the subspecies doylei strains contained a 3·4-MDa plasmid. These analyses showed that catalase-negative C. jejuni subsp. jejuni were genomically distinguishable from C. jejuni subsp. doylei as were Lior biotypes within subsp. jejuni . Ability to produce catalase is not a feature common to all C. jejuni strains, and our results confirm that some strains of subspecies jejuni may be negative in that character although typical in other respects. DNA pattern heterogeneity was consistent with serological differences between strains.     

Evaluation of a selective enrichment technique for the isolation of Campylobacter pylori

To cultivate Campylobacter pylori from contaminated biopsy specimens, Brucella broth was supplemented with 10% fetal calf serum, 1% Vitox, 1000 units/ml polymyxin B sulfate, 10 micrograms/ml vancomycin, and 2 micrograms/ml amphotericin B. Pseudomonas aeruginosa, Candida albicans, and Enterococcus fecalis were cocultivated with C. pylori. All four strains of C. pylori were recoverable at 24 h. When 21 C. pylori strains were studied in pure culture, 86% grew in the selective enrichment medium. In a clinical study, the selective enrichment technique resulted in isolation of C. pylori from 50% of patient samples, compared with isolation from only 36% of samples with agar cultivation. The selective enrichment technique may be more sensitive than techniques currently employed to isolate C. pylori from gastric tissue.     

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.     

Isolation and characterization of a novel catalase-negative, urease-positive Campylobacter from cattle faeces

Forty-four strains of a phenotypically unique Campylobacter were isolated from the faeces of 26 of 45 cows in a single herd. Isolation involved enrichment and membrane filtration onto blood agar or plating onto cefoperazone amphotericin teicoplanin agar. The strains exhibited phenotypic characteristics typical for Campylobacter species. However, they were unusual in that they produced urease and copious H₂S in triple sugar iron (TSI) medium, but did not produce catalase. They did not grow aerobically. None of the strains grew on modified cefoperazone charcoal deoxycholate agar (mCCDA). Macrorestriction profiles of chromosomal DNA were prepared for 15 strains using pulsed-field gel electrophoresis (PFGE). Twelve of 15 profiles were identical and all appeared to be closely related. These catalase-negative, urease-positive campylobacters (CNUPC) represent a group not previously reported. Their sensitivity to antibiotics normally used in selective media for campylobacters might explain why they have not previously been encountered. Their ecological significance and importance with respect to human and animal disease remain to be assessed.     

Dissemination of fluoroquinolone-resistant Campylobacter spp. within an integrated commercial poultry production system

While characterizing the intestinal bacterial community of broiler chickens, we detected epsilon-proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69:6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis. The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli. These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks.