Subspecies characterization of urease-positive thermophilic Campylobacter (UPTC) isolated from shellfish employing modified flagellin (flaA) restriction fragment length polymorphism (RFLP) typing.

ABSTRACT Shellfish including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis), have previously been described as an important source of thermophilic campylobacters, with the potential of causing acute bacterial gastroenteritis in humans. Previous genotyping studies employing the polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) typing, based on the flagellin (flaA) gene have been unable to generate an amplicon for the urease-positive thermophilic Campylobacter (UPTC), which are the predominant taxa associated with shellfish, largely caused by sequence diversity between the UPTC group and C. jejuni. Hence the aim of this study was to develop a modified PCR-RFLP genotyping assay, employing polymorphisms within the flagellin (flaA) gene of UPTC organisms, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from natural marine environments. A novel primer pair (UPTC flaF/UPTC flaR) was designed based on conserved regions within the flaA gene locus of UPTC organisms to generate a 1,358 bp amplicon for all UPTC organisms tested. RFLP analysis with DdeI in combination with computational analysis of genetic relatedness using BioNumerics software demonstrated the presence of four distinct flaA genotypes, among the seven UPTC isolates. In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences that may be successfully applied to examine subspecies relatedness of UPTC organisms from natural environments, including shellfish.

KEY WORDS: Campylobacter jejuni, DNA, flagellin (flaA), genotyping, PCR, shellfish, urease-positive thermophilic Campylobacter (UPTC)

INTRODUCTION

Thermophilic Campylobacter spp., including C. jejuni, C. coli and C. lari, are the most common cause of acute bacterial gastroenteritis in the developed Western world (Altekruse & Tollefson 2003). In Northern Ireland, laboratory confirmed isolates account for an approximate annual total of 800-1,000 cases, representing an attack rate of 59 cases per 100,000 individuals (Anon, 2002). In general, most Campylobacter infections are believed to be transmitted zoonotically to humans from various animal reservoirs, including poultry, cattle, pigs and from household pets, including cats and dogs, although the epidemiology and routes of transmission are still not completely understood. In addition, various reports have described the presence of thermophilic Campylobacter in shellfish, including oysters (Crassostrea gigas), cockles (Cerastoderma edule) and mussels (Mytilus edulis) (Wilson & Moore 1996, Endtz et al. 1997). Given that shellfish may be cultured and harvested in marine waters that are contaminated with agricultural run-off or with human sewage, shellfish concentrate fecal pathogens, including campylobacters mainly in their gill tissues and such produce may be a hazard to public health, particularly if (1) they are consumed raw; (2) are eaten following only a partial cooking or (3) are allowed to cross-contaminate other foods and utensils in the kitchen, where they are being prepared.

Various previous studies have demonstrated problems in the genotyping of the most common form of Campylobacter in shellfish, mainly the urease-positive thermophilic Campylobacter (UPTC) (Moore et al. 2003, Sekizuka et al. 2004), whereby these isolates failed to be typed using conventional flagellin (flaA)-based restriction fragment length polymorphism (RFLP) analysis, as described originally by Nachamkin et al. (1993). This inability to generate a PCR amplicon with the UPTC organisms was most likely to be caused by significant mutations in the flagellin gene structure in the UPTC organisms, compared with C. jejuni, from where the PCR primers were originally designed.

Hence, it was the aim of this study to develop a modified PCR-RFLP genotyping assay, using polymorphisms within the flagellin (flaA) gene of UPTC organisms as an epidemiological marker, which would now allow the successful amplification and typing of previously nontypable UPTC isolates obtained from shellfish and environmental water sources.

MATERIALS AND METHODS

Design of Modified flaA Primers

Two novel PCR primers were designed and designated as UPTC flaF(forward primer) and UPTC flaR (reverse). The flaA gene sequences of six UPTC isolates, which had been recently published, including UPTC AB07058, AB073915, AB073916, AB073917, AB073918 and AB080202 (GenBank Accession number), were aligned employing the Clustal alignment tool in combination with DNASTAR software (DNASTAR Inc, Wisconsin, USA). Conserved sites were selected (Fig. 1) and contained the following sequences UPTC flaF 5'-ATT AAC ACA AAT GGT GCA TCT TTA AAT G-3', corresponding to base positions 10-37 in relation to UPTC AB080202 and UPTC flaR 5'-CTC TAA TTT GTG ATT CAG CAG CTT TAA CAT T-3', corresponding to base positions 1357-1327 in relation to UPTC AB080202. Amplification using these primers was estimated to generate a PCR amplicon of approximately 1,358 bp in length.

[FIGURE 1 OMITTED]

Source of UPTC Isolates Used in This Study

Seven isolates belonging to the UPTC biovar of C. lari were used in this study, including four wildtype isolates and three reference isolates, obtained from different environments and in different countries, as detailed in Table 1. In addition, C. jejuni ATCC33560 was also used in this study.

DNA Extraction

All isolates were cultured under microaerophilic conditions (5% v/v C[O.sub.2]) for 48h on Butzler selective medium, and their species designation was confirmed, as described previously (Moore et al. 2003). Bacterial genomic DNA was extracted in accordance with the method of Harrington et al. (1999) and resulting nucleic acid (DNA) was quantified and adjusted to yield a working concentration of approximately 1.0 [micro]g/[micro]L. Extracted DNA was stored frozen at -20[degrees]C until used and at 4[degrees]C thereafter.

PCR Amplification and Restriction Fragment Length Polymorphism (RFLP) Analysis

After PCR optimization, amplification was performed on all seven UPTC isolates, as well as with the C. jejuni isolate, as detailed in Table 1. Reaction mixes (25 [micro]L) were set up as follows:-10 mM Tris-HC1, pH 8.3, 50 mM KC1, 2.5 mM Mg[Cl.sub.2], 200 [micro]M (each) dATP, dCTP, dGTP and dTTP; 1.25U of Taq DNA polymerase (Takara Corp., Tokyo, Japan), 10 pmol (each) of the forward and reverse primers (i.e., UPTC flaF/UPTC flaR) as described above and 1 [micro]L of DNA template. The reaction mixtures after a "hot start" were subjected to the following optimized thermal cycling parameters in a Perkin Elmer 9700 thermocycler: 96[degrees]C for 3 min followed by 40 cycles of 96[degrees]C for 1 min, 58.5[degrees]C for 1 min, 72[degrees]C for 2.5 min, followed by a final extension at 72[degrees]C for 10 min. During each run molecular grade water was included randomly as negative controls. After amplification, aliquots (15 [micro]L) were removed from each reaction mixture and examined by electrophoresis (100 V, 60 min) in gels composed of 1.0% (w/v) agarose (L03, Takara Corp., Japan) in 0.5 x TBE buffer, stained with ethidium bromide (5 [micro]g/100 mL). Gels were visualized under UV illumination using a gel image analysis system (Atta Image Saver, Atta Corp., Japan).

A restriction map was prepared from analysis of the 1,358 bp flaA fragment to estimate the optimal restriction enzymes(s) to use to detect polymorphisms in this fragment, using MapDraw software (DNAStar Inc., Wisconsin, USA). After PCR amplication, resulting amplicons (10 [micro]L) were digested to completion with the restriction endonuclease Dde I (10U) (cleavage site sequence CTGCA[down arrow]G; Toboyo Co. Ltd., Osaka, Japan), at 37[degrees]C for 4 h in the manufacturer's (H) buffer at the recommended concentration. Digested DNA fragments (15 [micro]l) from the Dde I digestion were subjected to electrophoresis, as described earlier, with the exception that digested fragments were separated on a 2.5% (w/v) agarose gel and visualized, as detailed earlier.

Computer Estimation of Genetic Relatedness

Computer analysis of the RFLP banding patterns obtained was performed with the BioNumerics software package (Applied Maths, Kortrijk, Belgium). Initially, all RFLP banding patterns were normalized and all images were compatible with one another after normalization, and complete RFLP patterns were used for analysis. In general, bands were automatically assigned by the computer and were corrected manually after the original images were visually checked. Only clearly resolved bands were counted. The Pearson coefficient was used to analyze the similarities of the banding patterns. The unweighted pair group method with average linkages (UPGAMA) was used for cluster analysis and the cophenetic correlation coefficient for the whole dendrogram was calculated for estimation of the faithfulness of the cluster analysis, using the BioNumerics software.

RESULTS AND DISCUSSION

PCR amplification using the novel primer pair, UPTC flaF/ UPTC flaA, allowed the reproducible generation of a flaA amplicon of approximately the expected size (circa 1,358 bp) for all UPTC isolates examined (Fig. 2). A slightly larger amplicon (circa 1,500 bp) was generated for C. jejuni ATCC33560 (data not shown). Restriction map analysis with DdeI demonstrated the presence of at least five restriction sites within the PCR amplicon, yielding fragments at positions 82, 155, 644, 885 and 910. Subsequent restriction of PCR amplicons demonstrated the presence of at least three bands, which were grouped into four banding profiles (genotypes), as determined by analysis by the BioNumerics software (Fig. 3).

[FIGURES 2-3 OMITTED]

Marine bivalve molluscs such as mussels (Mytilus edulis), cockles (Laevicardium edula), scallops (Pecter maximus) and oysters (Crassostrea gagas) are grown commercially in in-shore waters around Northern Ireland with an annual production and market value of 288.4 tons and 527,733 [pounds sterling] respectively. These shellfish feed by filtering plankton and detritis from large volumes of sea water, thereby concentrating gastrointestinal pathogenic organisms found in such waters, including Campylobacter, Salmonella and enteric viruses in their digestive system (Wilson & Moore 1996), which may be important vectors in the transmission of disease. The association between ingestion of raw shellfish and enteric diseases such as typhoid has been recognized and more recently, there has been several reports of shellfish-associated gastroenteritis with strong evidence for Campylobacter spp. as the etiologic agent. Campylobacter spp. have been found routinely in West Coast shellfish beds in the States of California, Oregon and Washington (Abeyta et al. 1993). Sample types included oysters, cockles, sediment, seawater, freshwater (streams and tributaries), bird excreta and cattle manure. Campylobacter spp. in the marine environment primarily comes from wild birds (Kapperud & Rosef 1983), farm runoff, surface water (Carter et al. 1987) and sewage bypasses (Jones 2001).

Urease-positive thermophilic Campylobacter (UPTC), a microaerophilic and Gram-negative bacterium, is an organism only identified relatively recently in England (Bolton et al. 1985). After the original descriptions of UPTC appeared, isolates of UPTC were reported in France, Ireland and The Netherlands, and UPTC strains have also recently been found in Japan (Matsuda et al. 1996). The original strains were isolated from river water, sea water, mussels and cockles. In 1988, Megraud et al. reported the first isolation of UPTC from human clinical infection, where these organisms were isolated from an appendix as well as from human feces (Megraud et al. 1988).

This group of organisms has been frequently found as the most frequent Campylobacter found in shellfish and waters (Wilson & Moore 1996).

The understanding of sources and means of transmission of UPTC organisms is important to help elucidate the epidemiology of these organisms in shellfish and the marine environment, as well as their potential transmission to humans. Consequently a reproducible, sensitive and well-standardized typing scheme is critical in the successful discrimination of such strains. Hence to elucidate the sources and modes of transmission of these organisms, it is essential to use epidemiological typing methods that discriminate between different strains but which are reliable and reproducible in the recognition of similar strains.

This study was interested in the ability to genotype members of the UPTC group of organisms isolated in shellfish and was undertaken to develop a PCR-based method that would allow the differentiation between UPTC organisms at the subspecies level. Until now, employing the conventional PCR-RFLP (fla A) method, as described originally for the subspecies differentiation of clinical C. jejuni, has not been successful in its application with the UPTC organisms (Moore et al. 2003). Furthermore, because this method is widely used for other campylobacters, it was important to develop it further, so that it may be used successfully with this group of organisms. Previous inabilities to generate a PCR amplicon using the Nachamkin flaA primers suggested that the UPTC organisms had an altered flagellin gene arrangement compared with C. jejuni, with various mutations at the original primer sites. Subsequently, we have previously reported this altered flagellin gene rearrangements in UPTC, through the sequencing and analyses of the flaA gene of several UPTC wildtype and reference strains (Sekizuka et al. 2004). Hence, it was the aim of the current study to use this sequence data from UPTC organisms and design a modified assay that would allow the successful amplification of these organisms, helping facilitate their subsequent subspecies analyses.

After the development of a successful and reproducible PCR-RFLP system, employing the newly described primer pair (UPTC flaF/UPTC flaR), we wished to establish proof-of-principle for this technique in demonstrating the application of this method to a small collection of UPTC isolates obtained from the natural environment. Application of this technique to this small collection of isolates demonstrated genetic variability at the flaA locus between these isolates (Fig. 3). Shellfish growing in relatively shallow marine waters, including inshore waters, may be contaminated with campylobacters from several sources, in particular, effluent from agricultural run-off and human sewage. In addition, they may become contaminated because of fecal deposition of UPTC from wild birds, including members of the gull (Larus) family. In this study, genotypic analysis demonstrated the presence of the same genotype of UPTC in shellfish isolated from the natural environment, located approximately 10-30 kilometers from each other, with no sharing of the same watertable. This may suggest that gulls may have been responsible from the transmission of the same genotype of UPTC between the two sites and hence acted as a vector of transmission of this particular genotype.

In conclusion, this study describes a PCR-RFLP method, based on modified primers from UPTC flaA gene sequences, that may be successfully applied to examine subspecies relatedness and routes of transmission of UPTC isolated from shellfish, so that we are better informed as to how these become contaminated to suggest intervention controls that would help reduce the loading of these shellfish with campylobacters. Further work is now required to establish routes of transmission of UPTC organisms from the environment to shellfish employing this modified technique.

ACKNOWLEDGMENTS

The authors thank Dr. Colin Fleming and Mr. Brendan Moreland, Applied Plant Science Research Division, Department of Agriculture and Rural Development for Northern Ireland, for advice and guidance regarding gel documentation analysis. JEM was supported financially by a grant made available by Azabu University and the Japan Private School Foundation, as well as the Research & Development Office, Department of Health & Public Safety, Northern Ireland (Infectious Disease-Recognised Research Group [RRG] 9.9).

LITERATURE CITED

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Altekruse, S. F. & L. K. Tollefson. 2003. Human campylobacteriosis: a challenge for the veterinary profession. J. Am. Vet. Med. Assoc. 223: 445-452.

Anon. 2000. Review of Communicable Diseases. Ed. Smyth B. Communicable Disease Surveillance Centre 2002:73. (CDSC)

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Carter. A. M., R. E. Pacha, G. W. Clark & E. A. Williams. 1987. Seasonal occurrence of Campylobacter spp. in surface waters and their correlation with standard indicator bacteria. Appl. Environ. Microbiol. 53: 523-526.

Endtz, H. P., J. S. Vliegenthart, P. Vandamme, H. W. Weverink, N. P. van den Braak, H. A. Verbrugh & A. van Belkum. 1997. Genotypic diversity of Campylobacter lari isolated from mussels and oysters in The Netherlands. Int. J. Food Microbiol. 34:79-88.

Harrington, C. S., F. M. Thomson-Carter & P. E. Carter. 1999. Molecular epidemiological investigation of an outbreak of Campylobacter jejuni identifies a dominant clonal line within Scottish serotype HS55 populations. Epidemiol. Infect. 122:367-375.

Jones, K. 2001. Campylobacters in water, sewage and the environment. Symp. Ser. Soc. Appl. Microbiol. 30:68S-79S.

Kapperud, G. & O. Rosef. 1983. Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp. and Salmonella spp. in Norway. Appl. Environ. Microbiol. 45:375-380.

Matsuda, M., A. Kaneko, M. Fukuyama, T. Itoh, M. Shingaki, M. Inoue & J. E. Moore. 1996. First finding of unease-positive thermophilic strains of campylobacter in river water in the Far East, namely, in Japan, and their phenotypic and genotypic characterization. J. Appl. Bacteriol. 81:608-612.

Megraud, F., D. Chevrier, N. Desplaces, A. Sedallian & I. L. Guesdon. 1988. Urease-positive thermophiic Campylobacter (Campylobacter lari variant) isolated from an appendix and from human faeces. J. Clin. Microbiol. 26:1050-1051.

Moore, J. E., A. Canney, T. Stanley, D. R. A. Wareing, A. Kaneko, L. Russell, B. C. Millar, P. G. Murphy & M. Matsuda. 2003. Phenotypic and genotypic characterization of urease-positive thermophilic Campylobacters (UPTC) isolated from shellfish, Int. J. Food Sci. Tech. 38:735-739.

Nachamkin, I., K. Bohachick & C. M. Patton. 1993. Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis. J. Clin. Microbiol. 31:1531-1536.

Sekizuka, T., K. Seki, T. Hayakawa, J. E. Moore, O. Murayama & M. Matsuda. 2004. Molecular cloning, nucleotide sequencing, and characterization of the flaA gene from strains of urease-positive thermophilic Campylobacter (UPTC). Res. Microbiol. 155:185-191.

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JOHN E. MOORE, (1)* MOTOO MATSUDA, (2) TSUYOSHI SEKIZUKA, (2) SEAMUS FANNING, (3) OHOSHI MURAYAMA, (2) TAEKO YOKOI, (2) SHIZUKO KAGAWA, (2) KENTARO NAGANO, (2) HIROMI SHIMURA, (2) MIYUKI WATABE, (1) YURIKO NAGANO, (1) KAORI USUI, (2) RIE IMAMAKI, (2) TAKAO ARITOMI, (2) CHISATO HARADA, (2) HARUNA IIDA, (2) NAOMI MITSUHASHI, (2) TAKESHI MIYATAKE, (2) MAKOTO SHIGEMATSU, (2) JULURI R. RAO, (4) COLM J. LOWERY, (5) BEVERLEY C. MILLAR, (1) JAMES S. G. DOOLEY (5) AND PAUL J. ROONEY (1)

(1) Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland, BT9 7AD; (2) Laboratory for Molecular Biology, School of Environmental Health Science, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan; (3) Faculty of Veterinary Medicine. University College Dublin, Belfield, Dublin 4, Ireland; (4) Applied Plant Science Research Division, Agri-Food and Biosciences Institute, Newforge Lane, Belfast, Northern Ireland, BT9 5PX; (5) School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co. Londonderry, Northern Ireland, BT52 1SA.

* Corresponding author. E-mail: [e-mail]

TABLE 1.

Isolate       Campylobacter
Reference       Organism        Source       Country

 15           UPTC *          Mussel        N. Ireland
145           UPTC            Mussel        N. Ireland
182           UPTC            Sea water     N. Ireland
CF89-12       UPTC            River water   Japan
NCTC12892     UPTC            River water   England
NCTC12895     UPTC            Mussel        England
NCTC12896     UPTC            Mussel        England
ATCC33560     C. jejuni       Bovine        USA

* UPTC, urease-positive thermophilic Campylobacter.