Campylobacter Sputorum Research Articles

Triplex qPCR assay for Campylobacter jejuni and Campylobacter coli monitoring in wastewater

Campylobacter spp. is one of the most frequent pathogens of bacterial gastroenteritis recorded worldwide. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the two major disease-associated species, accounting for >95 % of infections, and thus have been selected for disease surveillance. Monitoring temporal variations in pathogen concentration and diversity excreted from community wastewater allows the early detection of outbreaks. Multiplex real-time/quantitative PCR (qPCR) enables multi-target quantification of pathogens in various types of samples including wastewater. Also, an internal amplification control (IAC) is required for each sample when adopting PCR-based methods for pathogen detection and quantification in wastewater to exclude the inhibition of the wastewater matrix. To achieve reliable quantification of C. jejuni and C. coli towards wastewater samples, this study developed and optimized a triplex qPCR assay by combining three qPCR primer-probe sets targeting Campylobacter jejuni subsp. jejuni, Campylobacter coli, and Campylobacter sputorum biovar sputorum (C. sputorum), respectively. This triplex qPCR assay not only can directly and simultaneously detect the concentration of C. jejuni and C. coli in wastewater but also can achieve the PCR inhibition control using C. sputorum primer-probe set. This is the first developed triplex qPCR assay with IAC for C. jejuni and C. coli, to be used in the wastewater-based epidemiology (WBE) applications. The optimized triplex qPCR assay enables the detection limit of the assay (ALOD100%) and wastewater (PLOD80%) as 10 gene copy/μL and 2 log10 cells/mL (2 gene copies/μL of extracted DNA), respectively. The application of this triplex qPCR to 52 real raw wastewater samples from 13 wastewater treatment plants demonstrated its potential as a high-throughput and economically viable tool for the long-term monitoring of C. jejuni and C. coli prevalence in communities and the surrounding environments. This study provided an accessible methodology and a solid foundation for WBE-based monitoring of Campylobacter spp. relevant diseases and paved the road for future WBE back-estimation of C. jejuni and C. coli prevalence.

Oddziaływanie olejku cyprysowego (Cupressi aetheroleum) na bakterie mikroaerofilne

Introduction. The evergreen cypress (Cupressus sempervirens L.) belongs to the cypress family (Cupressaceae). It is considered a medicinal plant, and extracts from leaves, fruits and seeds are used in many ailments. The essential oil obtained from young twigs and cones of evergreen cypress contains, among others: α-pinene, δ3-carene, limonene, α-terpinene, β-myrcene, cedrol, β-pinene, δ-caryophyllene and β-caryophyllene, has antimicrobial activity and is recommended for external use. Aim. Testing the activity of cypress oil against microaerophilic bacteria occurring in the oral cavity. Material and methods. The microaerophilic bacteria for the study were isolated from the oral cavity They belonged to the species: Aggregatibacter actinomycetemcomitans, Campylobacter sputorum, Eikenella corrodens and Rothia dentocariosa. The date obtained 5 reference strains to. The susceptibility (MIC) was determined by means plate dilution technique in Brucella agar. The concentrations of cypress oil (Avicenna-oil, Wrocław) were: 5.0, 7.5, 10.0, 15.0 and 20.0 mg/ml. The inoculums containing 105 microorganisms in 1 ml were seeded with Steers replicator. Incubation performed in microaerophilic conditions in anaerobic jar with CAMPY Pak, BBL and standards strains in anaerobic atmosphere in anaerobic jars, in 37°C for 48 hours. The MIC was interpreted as the lowest concentration of cypress oil that completely inhibited the growth of tested microaerophilic bacteria. Results. The results indicated that the cypress oil was the most active in concentrations ≤ 5.0-7.5 mg/ml against 42% tested bacteria. Another concentrations in ranges 10.0-15.0 mg/ml was inhibited growth 26% strains. But 32% strains were susceptible on concentration 20.0 mg/ml and more. Tested cypress oil was the most active against strains from genus Rothia dentocariosa (MIC < 5.0-7.5 mg/ml). The genus Aggregatibacter actinomycetemcomitans was the lowest sensitive. The growth of this strains was inhibited in concentrations in ranges 5.0-≥ 2.0 mg/ml. Cypress oil was the most active at concentrations < 5.0-7.5 mg/ml against 47% of the assessed microaerophilic bacteria. Concentrations in the range of 10.0-15.0 mg/ml inhibited the growth of 26% of microorganisms. In contrast, 32% of the strains were sensitive to concentrations of 20.0 mg/ml and more. The tested cypress oil showed the highest activity against strains of the Rothia dentocariosa species (MIC < 5.0-7.5 mg/ml). Aggregatibacter actinomycetemcomitans was characterized by the lowest sensitivity. The growth of these bacteria was inhibited by concentrations of 5.0-≥ 20.0 mg/ml. Conclusions. Cypress oil was characterized by moderate activity against the tested bacteria growing in microaerophilic conditions, and the strains of the Rothia dentocariosa species showed the highest sensitivity to this oil.

Ocena aktywności olejku tatarakowego (Calami aetheroleum) wobec bakterii tlenowych i mikroaerofilnych

Introduction. Calamus (Acorus calamus L) grows in South and North America and Asia, it reached Europe with the invasions of the Tatars. It is a perennial with strongly branched, creeping, aromatic rhizome, strongly elongated, sword-shaped, pointed leaves. Reaches a height of up to 1 m. It has a cob-shaped inflorescence with green-yellow flowers. The fruits are small red berries, but in our climate calamus does not bear fruit. The extracts and essential oil obtained from the plant contain: ?- and ?-asarone, cyperenol, cyperol, acorin, acoritin, cyperenone, isoazarone, 2,2,5,5-tetramethyl-3-hexanol, galgavin, sacuranin, retusin, isoeugenol, elemicin, safrole, geranyl acetate, spathulenol, borneol, eugenol, linalool, camphor and linoleic, polmitinic and palmitoleic acid. Both the extract and the essential oil have antimicrobial properties. Aim. The investigation activity of calamus oil on aerobic and microaerophilic bacteria. Material and methods. The aerobic and microaerophilic bacteria were isolated from oral cavity and upper respiratory tract. The strains belonging to following genera: Staphylococcus (7), Enterococcus (4), Corynebacterium (2), Acinetobacter (3), Citrobacter (2), Escherichia (4), Klebsiella (2), Pseudomonas (5), Serratia (2), Aggregatibacter actinomycetemcomitans (11), Campylobacter (3), Eikenella (4), Rothia (2) and 7 reference strains were used. Investigation for aerobic bacteria was carried out in Mueller-Hinton agar which contain 2.5-20.0 mg/ml calamus oil (Semifarm) and incubation was carried out in 37°C for 24 hrs and for microaerophilic bacteria in range 0.12-2.0 mg/ml in Brucella agar with 5% sheep blood, and was incubated in anaerostats (CAMPY Pak, BBL) in 37°C for 48 hrs. The inoculums contained 10 5 CFU per spot. The lowest dilution of calamus oil that completely inhibited the growth of the tested bacteria was taken as the MIC. Results. The oil (10.0-15.0 mg/ml) inhibited the grows 4 strains from Staphylococcus aureus genus. MIC for remaining cocci were 15.0-? 20.0 mg/ml. The activity of calamus oil in the case of Gram-negative sticks turned out to be lower (15.0-? 20.0 mg/ml). The oil was more effective against microaerophilic bacteria (MIC 1.0-? 0.12 mg/ml). The strains from Campylobacter genus characterized the most susceptibility to oil (MIC ? 0.12-0.25 mg/ml). Until 81% of strains from Aggregatibacter actinomycetemcomitans genus was susceptible in concentrations ? 0.12-0.25 mg/ml. The rods from Eikenella and Rothia genus turned out to be sensitive to the concentration of the oil in the range of ? 0.12-1.0 mg/ml. Conclusions. The Gram-positive aerobic bacteria were most susceptible to calamus oil then Gram-negative rods. The oil was highly effective against microaerophilic bacteria of the Campylobacter sputorum species. Calamus oil showed higher activity against microaerophilic bacteria compared to aerobic microorganisms.

Species distribution, genetic diversity and antimicrobial susceptibility of Campylobacter isolates recovered from the preputial cavity of healthy rams in Turkey.

Campylobacter sp. are important causes of reproductive disease in ruminants worldwide. Although healthy bulls are well-known carriers for infection of cows, the role of rams as a potential source for infecting ewes is unclear. This study aimed to determine prevalence, species distribution, genetic diversity and antimicrobial susceptibility profiles of Campylobacter sp. isolated from the preputial cavity of healthy rams. The material of this prospective study comprised 191 swab samples taken from the preputial cavity of healthy rams. Enrichment and membrane filtration were employed for the isolation of Campylobacter. Presumptive isolates were confirmed as Campylobacter by phenotypic and molecular tests. 16S rRNA gene sequence analysis was used for the definitive identification of the isolates at species level, and genotyping was performed using pulsed-field gel electrophoresis (PFGE). The susceptibility of the Campylobacter sp. isolates to various antibiotics was determined by the disk diffusion test. In all, 27 of the 191 (14·13%) swab samples were found to be positive for Campylobacter sp. (28 isolates were recovered in total). Per phenotypic and genotypic analyses, one isolate was identified as Campylobacter mucosalis and the remaining 27 isolates were identified as Campylobacter sputorum bv. faecalis. The PFGE analysis of the C. sputorum biovar faecalis isolates produced 17 clusters and 24 different pulsotypes, indicating high genetic heterogeneity. All 28 isolates were found to be susceptible to all of the antibiotics tested. Healthy rams may be an important reservoir of different Campylobacter species in the preputium. This study demonstrated for the first time that healthy rams can carry different Campylobacter sp. including genetically diverse C. sputorum bv. faecalis and C. mucosalis in the preputial cavity. Further investigation on the potential implication of this finding on sheep reproductive health (e.g. infectious infertility, and abortion) and overall epidemiology of Campylobacter may be warranted.

Complete Genome Sequences of the Campylobacter fetus subsp. venerealis, Campylobacter lari subsp. concheus, Campylobacter sputorum bv. sputorum, and Campylobacter volucris Type Strains.

Campylobacter spp. are recovered from a wide variety of sources, including birds, livestock, shellfish, and human clinical samples. We present here the complete genomic data for the type strains of Campylobacter fetus subsp. venerealis, Campylobacter lari subsp. concheus, Campylobacter sputorum bv. sputorum, and Campylobacter volucris.

Genomic Evidence for the Emergence and Evolution of Pathogenicity and Niche Preferences in the Genus Campylobacter

The genus Campylobacter includes some of the most relevant pathogens for human and animal health; the continuous effort in their characterization has also revealed new species putatively involved in different kind of infections. Nowadays, the available genomic data for the genus comprise a wide variety of species with different pathogenic potential and niche preferences. In this work, we contribute to enlarge this available information presenting the first genome for the species Campylobacter sputorum bv. sputorum and use this and the already sequenced organisms to analyze the emergence and evolution of pathogenicity and niche preferences among Campylobacter species. We found that campylobacters can be unequivocally distinguished in established and putative pathogens depending on their repertory of virulence genes, which have been horizontally acquired from other bacteria because the nonpathogenic Campylobacter ancestor emerged, and posteriorly interchanged between some members of the genus. Additionally, we demonstrated the role of both horizontal gene transfers and diversifying evolution in niche preferences, being able to distinguish genetic features associated to the tropism for oral, genital, and gastrointestinal tissues. In particular, we highlight the role of nonsynonymous evolution of disulphide bond proteins, the invasion antigen B (CiaB), and other secreted proteins in the determination of niche preferences. Our results arise from assessing the previously unmet goal of considering the whole available Campylobacter diversity for genome comparisons, unveiling notorious genetic features that could explain particular phenotypes and set the basis for future research in Campylobacter biology.

Construction and Expression of a Recombinant Urease Gene Cluster from Campylobacter Sputorum Biovar Paraureolyticus

Recombinant full-length urease gene cluster and seven 100% deletion recombinant variants of urease subunits genes, (ureG, ureH, ureA, ureB, ureC, ureE and ureF) were constructed in vitro from the Campylobacter sputorum biovar paraureolyticus LMG17591 strain and expressed in Escherichia coli JM109 cells. A urease-positive reaction (1.885 μmol/min/mg protein) in the log-phase cultured E. coli cells transformed with pGEM-T vector carrying the recombinant full-length urease genes cluster was detected. Among the seven 100% deletion recombinant variants, each of the ureG-, ureH(D)-, ureA-, ureB-, ureC-, ureE- and ureF-deletion variants showed no change in assay of the urease reaction, and similarly as in the E. coli cell lysate with pGEM-T vector only. Recombinant full-length urease gene cluster and 100% deletion recombinants of the ureE gene in the transformed and log-phase cultured E. coli cells from the C. sputorum showed positively accelerated urease activities when cultured in the medium containing NiCl2 (750 μmol/L), but no activity was accelerated in the C. sputorum cultured in NiCl2. In addition, thiourea (20 mmol/L) completely inhibited urease activities from all C. sputorum examined. The putative recombinant urease subunits A and C were immunologically identified by Western blot analysis with polyclonal anti-urease α (A) and β (B), raised against Helicobacter pylori.

Isolation of Campylobacter from Feral Swine (Sus scrofa) on the Ranch Associated with the 2006 Escherichia coli O157:H7 Spinach Outbreak Investigation in California

We report the isolation of Campylobacter species from the same population of feral swine that was investigated in San Benito County, California, during the 2006 spinach-related Escherichia coli O157:H7 outbreak. This is the first survey of Campylobacter in a free-ranging feral swine population in the United States. Campylobacter species were cultured from buccal and rectal-anal swabs, colonic faeces and tonsils using a combination of selective enrichment and antibiotic-free membrane filtration methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS, Bruker Daltonics, Inc., Billerica, MA, USA) was used to identify species followed by confirmatory multiplex PCR or 16S rRNA sequencing. Genetic relatedness of Campylobacter jejuni and Campylobacter coli strains was determined by multilocus sequence typing (MLST) and porA allele sequencing. Altogether, 12 (40%) of 30 feral swine gastrointestinal and oral cavity specimens were positive, and six species were isolated: Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalsis, Campylobacter jejuni, Campylobacter lanienae and Campylobacter sputorum. Campylobacter jejuni subtypes were closely related to MLST sequence type 21 (ST-21) and had identical porA sequences. Campylobacter coli subtypes were unrelated to isolates in the pubMLST/porA database. This feral swine population lived in close association with a 'grassfed' beef cattle herd adjacent to spinach and other leafy green row crop fields. The findings underscore the importance of protecting raw vegetable crops from faecal contamination by wild or feral animals. The study also illustrates a potential risk of Campylobacter exposure for hunters during handling and processing of wild swine meat.

Emerging ThermotolerantCampylobacterSpecies in Healthy Ruminants and Swine

Campylobacters other than Campylobacter jejuni or C. coli were isolated in 35% of 343 farms recently analyzed in northern Spain. This study was aimed at identifying at the species level the 120 isolates collected (21 ovine, 52 beef cattle, 44 dairy cattle, and 3 porcine) by species-specific polymerase chain reaction and 16S rRNA gene sequencing analysis. Thus, five species were identified: Campylobacter hyointestinalis (90 isolates), Campylobacter lanienae (12), Campylobacter fetus subsp. fetus (10), Campylobacter lari (1), and Campylobacter sputorum (1). Ambiguous results were obtained for six isolates. Phylogenetic analyses of the 16S rRNA gene sequence placed three of them (cattle isolates) as an intermediate clade between C. hyointestinalis subsp. hyointestinalis and C. fetus, two ovine isolates formed a new clade clustering with Campylobacter concisus despite sharing higher similarity with Campylobacter mucosalis, and one porcine isolate shared similarly high homology with C. lanienae and C. hyointestinalis subsp. lawsonii. C. hyointestinalis was the predominant species, particularly in cattle, but it was also isolated from sheep and swine. C. lanienae was only found in sheep, C. fetus in cattle and sheep, and C. lari in a single dairy cattle farm. Although previously reported, the isolation of C. lari from cattle is not common, and this is the first report of C. lanienae and C. hyointestinalis in sheep. This study demonstrated the wide distribution in livestock of several emerging zoonotic Campylobacter species and provided valuable information on their host animal reservoirs.

Molecular analysis and characterization of a urease gene operon from Campylobacter sputorum biovar paraureolyticus

When recombinant plasmid DNA from a genomic DNA library and inverse PCR products of Campylobacter sputorum biovar paraureolyticus LMG17591 strain were analyzed, an approximate 6.5-kb pair region, encoding a urease gene operon, was identified. Within the operon, seven closely spaced and putative open reading frames for ureG, ureH(D), ureA, ureB, ureC, ureE, and ureF were detected in order. A possible overlap was detected between ureG and ureH(D), ureH(D) and ureA, and ureE and ureF. In addition, two putative promoter structures, probable ribosome-binding sites and a putative ρ-independent transcriptional terminator structure were identified. The urease gene operon transcription in the cells was confirmed by the reverse transcription-PCR analysis. A neighbor-joining tree constructed based on the nucleotide sequence information of urease genes showed that C. sputorum biovar paraureolyticus formed a cluster with Arcobacter butzleri, urease-positive thermophilic Campylobacter and some Helicobacter spp., separating those from the other urease-producing bacteria, suggesting a commonly shared ancestry among these organisms.

Occurrence and characterisation of intervening sequences (IVSs) within 16S rRNA genes from two atypical Campylobacter species, C. sputorum and C. curvus

A polymerase chain reaction (PCR) method was carried out on 21 isolates of atypical Campylobacter sputorum (n=14) and C. curvus (n=7) using a primer pair to amplify the helix 11 region within 16S ribosomal RNA (rRNA) gene sequences. Following sequencing and alignment analysis, 14 C. sputorum (100%) and six C. curvus (86%) isolates were shown to carry intervening sequences (IVSs) in this region. Interestingly, the nucleotide sequences of all the IVSs were identical among the 14 C. sputorum isolates (n=5 C. sputorum biovar [bv] paraureolyticus; n=5 bv fecalis; n=4 bv sputorum). In addition, two different nucleotide lengths and sequences of IVSs were identified among the six C. curvus isolates. On the first prediction of the secondary structure model of the IVSs in 16S rRNA genes, stem and loop structures were identified. In the purified RNA fractions from the 20 Campylobacter isolates carrying IVSs, no 16S rRNA was evident. Instead, other smaller RNA fragments were identified. Thus, the primary 16S rRNA transcripts may have been fragmented in the 20 isolates. This is the first demonstration of atypical C. sputorum and C. curvus isolates carrying IVSs in the helix 11 region in 16S rRNA genes.

Prevalence of Campylobacter species in apparently healthy goats in Sokoto state (Northwestern) Nigeria

A total of 1312 rectal swabs (feacal) samples were collected from apparently healthy goats across Sokoto state and processed for Campylobacter organisms. 264 (20.1%) of the samples were positive and yielded 272 Campylobacter species.Campylobacter species isolated in this study were Campylobacter jejuni 169(62.1%),Campylobacter coli 58(21.3%), Campylobacter lari 24(8.8%), Campylobacter upsaliensis 13(4.8%) and Campylobacter sputorum 8(3.0%). Biotyping of the isolates indicated that C. jejuni biotype I (52.6%) and C. coli biotype II (82.8%) were the most common biotypes while all the C. lari isolates were biotype I. The isolation of Campylobacter organisms and the identification of C. jejuni biotype I and C. colibiotype II from goats in this study is a clear indication of the presence ofCampylobacter in goats in Sokoto state. Campylobacter organisms must be considered as potential agent of enteritis and abortion in goats as well as a serious public health problem. Key words: Biotypes, Campylobacter, Campylobacter jejuni, Campylobacter coli,Nigeria, prevalence, goats.

Differentiation of Campylobacter species by AFLP fingerprinting

The fluorescent amplified fragment length polymorphism (AFLP) fingerprinting method was tested for its ability to identify and subtype the most important Campylobacter species found in veterinary infections. Sixty-nine reference strains and 19 clinical isolates of Campylobacter jejuni subsp. jejuni, Campylobacter jejuni subsp. doylei, Campylobacter upsaliensis, Campylobacter coli, Campylobacter lari, Campylobacter fetus subsp. fetus, C. fetus subsp. venerealis, Campylobacter hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii, Campylobacter mucosalis, Campylobacter helveticus and Campylobacter sputorum were subjected to analysis. The topology of the dendrogram obtained by numerical analysis of the AFLP profiles did not reflect the phylogenetic relationships as derived from 16S rDNA sequence comparison. However, except for C. lari, AFLP analysis grouped the strains that belonged to the same genomic species into distinct clusters. C. lari strains were separated into two distinct AFLP groups, which corresponded with nalidixic-acid-sensitive and -resistant variants of C. lari. These results correlated with data from whole-cell protein profiling. Within C. jejuni, C. hyointestinalis and C. fetus, strains could be identified at the subspecies level. AFLP analysis also allowed the subtyping of most species at the strain level. It is concluded that AFLP analysis is a valuable tool for concurrent identification of campylobacters at the species, subspecies and strain levels. In addition, the data confirm and extend previous reports showing that C. lari is a heterogeneous species that may comprise multiple taxa.

Taxonomy of Campylobacter, Arcobacter, Helicobacter and related bacteria: current status, future prospects and immediate concerns.

The taxonomy of the genus Campylobacter has changed dramatically since its inception in 1963. At that time the genus comprised just two species. At present, taxa that were once assigned to Campylobacter may belong to one of over 50 species distributed among six genera. Most of these taxa belong to a phylogenetically distinct group referred to as either ribosomal RNA (rRNA) superfamily VI or the epsilon division of the class Proteobacteria. The taxonomic diversity of the group is matched by the diverse habitats in which they may be found, and by the wide range of diseases that they are associated with. Recognition of their clinical and economic importance has resulted in intense interest in the group, and the application of increasingly sophisticated isolation, detection and chemotaxonomic methods continues to elucidate new aspects of their biodiversity. However, despite the advances in new bacterial systematics, there remain a number of important issues concerning the classification of various campylobacterial taxa that require careful consideration. Ultimately, these issues are relevant to many working in the field of applied microbiology, including clinicians, veterinarians, epidemiologists and taxonomists. The purpose of this article is briefly to review the major developments in the taxonomy of Campylobacter from its inception to the present day; summarize the most recent changes in the field; analyse current topical issues of special relevance to applied microbiologists, including identification of the bacteria; and speculate on future prospects for campylobacterial taxonomy.

Campylobacter hominis sp. nov., from the human gastrointestinal tract.

Sequences of 16S rDNA of a novel campylobacter from faeces of healthy humans were previously shown to originate from a new taxon, 'Candidatus Campylobacter hominis', which could not be cultured. Since phylogenetic analysis suggested that anaerobic conditions might be required for growth, an isolation strategy was developed employing initial non-selective membrane filtration onto fastidious anaerobe agar. Campylobacters were then isolated from the resulting mixed microbial flora by a dilution strategy and/or by immunomagnetic separation with genus-specific polyclonal antibody. Isolates were identified by a genus and taxon-specific PCR assay, and 16S rDNA nucleotide sequence analysis was carried out. All isolates exhibited the typical Campylobacter characteristics of being non-fermentative, oxidase-positive, catalase-negative and Gram-negative. Unusually, however, they were straight rods lacking flagella. The 16S rDNA nucleotide sequence analysis, DNA and mol% G+C were consistent with a new Campylobacter species whose nearest phylogenetic neighbours were Campylobacter gracilis and Campylobacter sputorum. The unique species status of the isolates was further confirmed by taxonomic analysis of 47 phenotypic characteristics. The name Campylobacter hominis sp. nov. is proposed for the new species, the type strain of which is NCTC 13146T (= LMG 19568T).

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.

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.

Clonality of Campylobacter sputorum bv. paraureolyticus determined by macrorestriction profiling and biotyping, and evidence for long-term persistent infection in cattle.

Eighteen strains of Campylobacter sputorum bv. paraureolyticus (isolated over a 12-month period from seven dairy cows contained in a single herd) were examined by resistotyping, and macrorestriction profiling using pulsed field gel electrophoresis (PFGE). The resistotypes of these strains were identical, although repeat testing indicated resistance to metronidazole was not a reliable trait for typing purposes. Five SmaI-derived genotypes were identified among the 18 strains. In 5 of 7 cows, isolates obtained from the same animal, but from different time periods, were genotypically indistinguishable, indicating persistence of infection. Macrorestriction profiles of 5 strains representing the 5 SmaI genotypes and 8 other strains of C. sputorum from various sources, were prepared using 4 endonucleases (SmaI, SalI, BamHI and KpnI). The only other strain of C. sputorum bv. paraureolyticus examined (a Canadian isolate from human faeces), was found to have a SmaI macrorestriction profile identical with one of the five clones isolated from the cattle. Moreover, SalI and BamHI profiles of all bv. paraureolyticus strains were similar, while digestion with KpnI was not observed. By contrast, the seven strains of C. sputorum bv. sputorum yielded various macrorestriction profiles with all the enzymes used, and features distinguishing the two biovars studied could be identified. This study indicates that C. sputorum can persist in cattle for at least 12 months and exhibits a clonal population genetic structure.

The isolation and prevalence of campylobacters from dairy cattle using a variety of methods.

Faecal samples from 94 dairy cows and 42 calves in three different herds were examined by a variety of techniques for campylobacters. Cefoperazone amphotericin teicoplanin (CAT) agar, modified cefoperazone charcoal deoxycholate agar (mCCDA), Karmali agar, and membrane filtration onto blood agar, were used with and without enrichment in CAT broth. Seventy-nine percent of cattle in herd A carried campylobacters, compared with 40% and 37.5% of cattle in herds B and C, respectively. Most animals carried only one species of Campylobacter. Campylobacter hyointestinalis was isolated most frequently (32% animals positive) with Camp. fetus subsp. fetus and Camp. jejuni subsp. jejuni detected in 11% and 7% of animals, respectively. In addition, a novel biotype of Camp. sputorum was isolated from 60% of 47 cows tested in herd A. Direct plating detected only two of the total of 40 animals positive for campylobacter. Enrichment in CAT broth before membrane filtration onto blood agar or CAT agar were the most successful methods of plating. Campylobacter sputorum was isolated from CAT agar and blood agar but not from mCCDA or Karmali agar. Karmali agar incubated at 30 degrees C was especially effective for isolating Camp. fetus subsp. fetus.

Emended description of Campylobacter sputorum and revision of its infrasubspecific (biovar) divisions, including C. sputorum biovar paraureolyticus, a urease-producing variant from cattle and humans.

A polyphasic taxonomic study of 15 bovine and human strains assigned to the catalase-negative, urease-positive campylobacter (CNUPC) group identified these bacteria as a novel, ureolytic biovar of Campylobacter sputorum for which we propose the name C. sputorum bv. paraureolyticus: suitable reference strains are LMG 11764 (human isolate) and LMG 17590 (= CCUG 37579, bovine isolate). The present study confirmed previous findings showing that the salient biochemical tests used to differentiate C. sputorum bv. sputorum from C. sputorum bv. bubulus are not reproducible; and that the absolute validity of source-specific biovars of the species is questionable. A correlation between the results of numerical analysis of protein profiles and the reaction of strains in certain enzyme tests was, however, noted. Therefore, it is proposed that the infrasubspecific (biovar) divisions of C. sputorum should be revised to include bv. sputorum for catalase-negative strains; bv. fecalis for catalase-positive strains; and bv. paraureolyticus for urease-positive strains. Strains classified previously as bv. bubulus should be reclassified as bv. sputorum. The species description of C. sputorum is revised accordingly.