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1 SAC Veterinary Services, Drummondhill, Stratherrick Road, Inverness IV2 4JZ, UK
2 Health Protection Agency, National Collection of Type Cultures, Central Public Health Laboratory, London NW9 5HT, UK
3 Centers for Disease Control and Prevention, US Department of Health and Human Services, 1600 Clifton Road NE, Atlanta, GA 30333, USA
4 School of Food Biosciences, University of Reading, Whiteknights, Reading RG6 6AP, UK
5 School of Natural and Environmental Sciences, Coventry University, Priory Street, Coventry CV1 5FB, UK
6 Health Protection Agency, Helicobacter Reference Unit, Central Public Health Laboratory, London NW9 5HT, UK
7 University of Aberdeen, School of Biological Sciences, Lighthouse Field Station, Cromarty, Ross-shire IV11 8YJ, UK
Correspondence
Geoffrey Foster
g.foster{at}ed.sac.ac.uk
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Published online ahead of print on 24 September 2004 as DOI 10.1099/ijs.0.63147-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of NCTC 12927T is AJ620504.
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). In 1963, this organism was transferred to the newly proposed genus Campylobacter (Sebald & Véron, 1963). During the 1970s, a renewed interest in Campylobacter followed the recognition of Campylobacter jejuni and Campylobacter coli as a cause of diarrhoea in humans (Cooper & Slee, 1971; Dekeyser et al., 1972) and campylobacters soon became recognized as the commonest bacterial cause of enteritis (Griffiths & Park, 1990; Penner, 1988). In addition, the implementation of improved phylogenetic methods of analysis permitted closer taxonomic scrutiny of these organisms, resulting in several novel species, and led to the proposal of rRNA superfamily VI of the Proteobacteria as including the genera Campylobacter, Helicobacter and Arcobacter (Vandamme et al., 1991). According to On (2001), the genus Campylobacter contains 16 species with a further six subspecies. In the course of bacteriological investigations of rectal swabs from free-ranging seals and also following post-mortem examination of seals and cetaceans, four Campylobacter-like organisms were recovered from three seals and a porpoise. In this paper, we describe the cultural and biochemical characteristics of these bacteria and the results of a polyphasic taxonomic investigation.
Three Campylobacter-like organisms were isolated from rectal swabs collected from three common seals (Phoca vitulina) during a capture–release programme (Thompson et al., 1992). A fourth strain was recovered from a sample of small intestine taken from a harbour porpoise (Phocoena phocoena) carcass submitted under the Scottish Strandings Scheme. Isolation was made on Columbia blood agar base (Oxoid) supplemented with Blaser–Wang selective supplement (Oxoid) and 5 % (w/v) citrated sheep blood agar (CSBA) (Oxoid). Plates were incubated at 37 °C in a microaerobic atmosphere achieved by jar evacuation and filling with a prepared gas mixture containing N2/CO2/O2 (84 : 10 : 6). Culture plates were examined for growth after 2, 4 and 7 days incubation. Isolates were transferred to fresh citrated sheep blood agar plates for maintenance and characterization tests. The four strains were examined in a range of tests, the methods for most of which have been described by On & Holmes (1995), taking special note, where appropriate, of inoculum size as recommended in On & Holmes (1991). Acid production from carbohydrates was tested for in peptone yeast sugars. For motility determination, a blood-agar slope was inoculated with a broth suspension of the organism until a small amount of the inoculum remained at the base of the slope. Following overnight incubation, motility was determined from microscopic examination of a wet preparation of the broth at the base of the slope. Nitrate reduction was tested by adding Greiss–Ilosvay's reagents nos 1 and 2 (Merck) to a 24 h culture in nutrient broth no. 2 (Unipath) containing 1 % (w/v) KNO2. Antibiotic-susceptibility tests were carried out by disc diffusion using cephalothin (30 µg) and nalidixic acid (30 µg) discs (Oxoid).
16S rRNA genes of the isolates were amplified by a PCR using universal primers pA (sequence 5'-AGAGTTTGATCCTGGCTCAG-3', corresponding to positions 8–28 in Escherichia coli numbering) and pH (sequence 5'-AAGGAGGTGATCCAGCCGCA-3', positions 1542–1522 in E. coli numbering) and directly sequenced using a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems) and an automatic DNA sequencer (model 373A; Applied Biosystems). The closest known relatives of the novel isolates were determined by searching the GenBank database using FASTA (Lipman & Pearson, 1985). These sequences and those of other known related strains were retrieved from GenBank and aligned with the newly determined sequences using the program DNATools (Rasmussen, 1995). The resulting multiple sequence alignment was corrected manually and a distance matrix was calculated with the programs PRETTY and DNADIST (using the Kimura-2 correction parameter) (Felsenstein, 1989). A phylogenetic tree was constructed according to the neighbour-joining method with the program NEIGHBOR, and the stability of the groupings was estimated by bootstrap analysis (500 replications) using the programs DNABOOT, DNADIST, NEIGHBOR and CONSENSE (Felsenstein, 1989). For DNA–DNA hybridization, the bacterial cells were lysed and DNA was isolated and purified according to the method of Brenner et al. (1982). DNA from strain NCTC 12927T was labelled with [32P]dCTP by utilizing a nick translation kit (Invitrogen Life Technologies). This preparation was tested for reassociation to unlabelled DNA from the same strain and to the three other Campylobacter isolates as well as to DNAs from the type strains of Campylobacter lari, C. jejuni and C. coli. Relative binding ratios and divergence were calculated as described by Brenner et al. (1982). All reactions were done in duplicate at both the optimal temperature (50 °C) and the stringent temperature (65 °C). The clonality of the four Campylobacter-like organisms was investigated by amplified fragment length polymorphism (AFLP) fingerprinting for which DNA was extracted as previously described by Gibson et al. (1998). A single-enzyme AFLP (SAFLP) method was used in which the DNA was diluted to a concentration of 0·266 µg µl–1. The SAFLP method was carried out as described by Champion et al. (2002) except that PCR amplification was performed in a Sprint thermal cycler (Hybaid). Cluster analysis was performed with BIONUMERICS (Applied Maths), using the Dice correlation coefficient and the UPGMA clustering algorithm.
Four strains of Gram-negative, comma-shaped bacilli morphologically similar to Campylobacter were isolated from rectal swabs taken from three common seals and the intestinal contents of a porpoise. The organisms were catalase-positive, oxidase-positive and motile. Colonies of all the isolates, when grown on CSBA at 37 °C in a microaerobic atmosphere, were 0·75–1·0 mm in diameter, circular, entire, low-convex, smooth, shiny, grey, translucent, butyrous and easily emulsified. Swarming did not occur. Growth did not occur at either 25 or 42 °C, nor did it occur in aerobic or anaerobic conditions. Growth did occur on unsupplemented nutrient agar and in the presence of 1 % (w/v) glycine but not in the presence of 2 or 3·5 % (w/v) NaCl. Urease was not produced. Nitrates were reduced but nitrites were not reduced. The strains were non-saccharolytic and were resistant to 30 µg cephalothin ml–1 and 30 µg nalidixic acid ml–1. Hydrogen sulphide was produced in triple-sugar–iron (TSI) agar. Hippurate and indoxyl acetate were not hydrolysed.
To clarify the taxonomic interrelationships of the unidentified isolates, their 16S rRNA gene sequences were determined. The almost complete gene sequences of all four strains were elucidated and pairwise comparisons showed that all of the isolates were genetically highly related to each other, exhibiting 99·9–100 % sequence similarity (based on a comparison of approximately 1400 bases). The 16S rRNA gene sequence (>1400 nt) of a representative strain (NCTC 12927T) was subjected to searches of GenBank/EMBL, which confirmed that the unknown bacterium was phylogenetically most closely related to the genus Campylobacter (data not shown). A tree constructed using the neighbour-joining method and depicting the phylogenetic position of the unidentified organism within the genus Campylobacter is shown in Fig. 1. The unknown bacterium formed a distinct subline within the genus, associated with a subcluster of species that included C. jejuni subsp. jejuni and subsp. doylei, C. lari and C. coli. Pairwise sequence comparisons revealed similarities of 98·8, 98·3 and 97·6 %, respectively, with the aforementioned species. Other Campylobacter species displayed substantially lower levels of similarity (data not shown).
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. The four sea-mammal isolates displayed relative binding ratios >70 % under both optimal and stringent hybridization conditions, thereby demonstrating that they are members of a single genomic species. This was consistent with very low divergence (%D) values. By contrast, C. lari displayed a relative binding ratio of 61 % under optimum conditions and 33 % under stringent conditions, with a D value of 11·7 % with respect to the reference organism (strain NCTC 12927T). Significantly lower relative binding ratios and higher %D values were shown between the unknown bacterium and C. jejuni and C. coli (Table 1).
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, several groups were discerned within this species. The isolates recovered from marine mammals also resemble C. lari phenotypically. However, the novel species can be differentiated from C. lari by its inability to grow at 42 °C or in the presence of 2 % (w/v) NaCl. Therefore, on the basis of both phenotypic and molecular genetic evidence, we are of the opinion that the organisms from marine mammals merit classification as a novel species of the genus Campylobacter, for which the name Campylobacter insulaenigrae sp. nov. is proposed. Tests that serve to differentiate C. insulaenigrae from other members of the genus Campylobacter are shown in Table 2. It is pertinent to note that the four isolates of C. insulaenigrae were recovered from different animals and at different times. AFLP genetic profiling (Fig. 2) showed that the isolates were all genetically different from each other, thereby demonstrating that they represent different strains rather than a single clone.
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The following description of morphological and physiological characteristics is based on the results of studies of four strains. Gram-negative, motile, non-encapsulated, non-spore-forming, comma-shaped rods. Colonies on CSBA incubated in a microaerobic atmosphere at 37 °C for 48 h are 0·75–1·0 mm in diameter, circular, entire, low-convex, smooth, shiny, grey, translucent, butyrous and easily emulsified. Growth does not occur at 25 or 42 °C, or in aerobic or anaerobic atmospheres. Growth occurs in the presence of 1 % (w/v) glycine but not in the presence of 2 or 3·5 % (w/v) NaCl. Oxidase- and catalase-positive, but urease-negative. Nitrates are reduced, but nitrites are not reduced. H2S is produced in TSI agar, but hippurate and indoxyl acetate are not hydrolysed. Resistant to 30 µg cephalothin and 30 µg nalidixic acid ml–1.
The type strain is NCTC 12927T (=CCUG 48653T). Isolated from marine mammals.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Champion, O. L., Best, E. L. & Frost, J. A. (2002). Comparison of pulsed-field gel electrophoresis and amplified fragment length polymorphism techniques for investigating outbreaks of enteritis due to campylobacters. J Clin Microbiol 40, 2263–2265.
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