Multilocus sequence typing of Campylobacter jejuni isolates from New South Wales, Australia

Authors


L. Mickan, Infectious Diseases Laboratories Institute of Medical and Veterinary Science, P.O. Box 14, Rundle Mall Post Office, Adelaide, SA 5000, Australia.
E-mail: lance.mickan@imvs.sa.gov.au

Abstract

Aims:  Multilocus sequence typing (MLST) was used to examine the diversity and population structure of Campylobacter jejuni isolates associated with sporadic cases of gastroenteritis in Australia, and to compare these isolates with those from elsewhere.

Methods and Results:  A total of 153 Camp. jejuni isolates were genotyped. Forty sequence types (STs) were found, 19 of which were previously undescribed and 21 identified in other countries. The 19 newly described STs accounted for 43% of isolates, 16 of which were assigned to known clonal complexes. Eighty-eight percent of isolates were assigned to a total of 15 clonal complexes. Of these, four clonal complexes accounted for 60% of isolates. Three STs accounted for nearly 40% of all isolates and appeared to be endemic, while 21 STs were represented by more than one isolate. Seven infections were acquired during international travel, and the associated isolates all had different STs, three of which were exclusive to the travel-acquired cases. Comparison of serotypes among isolates from clonal complexes revealed further diversity. Eight serotypes were identified among isolates from more than one clonal complex, while isolates from six clonal complexes displayed serotypes not previously associated with those clonal complexes.

Conclusions:  Multilocus sequence typing is a useful tool for the discrimination of subtypes and examination of the population structure of Camp. jejuni associated with sporadic infections.

Significance and Impact of the Study:  This study highlights the genotypic diversity of Camp. jejuni in Australia, demonstrating that STs causing disease have both a global and a local distribution evident from the typing of domestically and internationally acquired Camp. jejuni isolates.

Introduction

Human campylobacteriosis, primarily caused by Campylobacter jejuni, is the most frequently notified gastrointestinal infection in Australia. The prevalence of notified cases in Australia was 116·5/100 000 population in 2003 (Miller et al. 2005). Consistent with other developed nations including the Netherlands and the United States, Campylobacter infections in Australia accounted for more than twice the number of notifications of Salmonella and Shigella combined (Communicable Diseases Network Australia 2003; Schouls et al. 2003). Although infection with Campylobacter has been associated with a variety of risk factors including handling and consumption of poultry products, raw or inadequately heat-treated milk, inadequately treated water and contact with domestic pets (Butzler 2004), the source of infection often remains unidentified (Adak et al. 1995). Most infections are considered to be sporadic, with outbreaks occurring only infrequently (Pebody et al. 1997).

Epidemiological investigations of Campylobacter infection have been hampered by the lack of a suitable, widely available typing method; although numerous phenotyping and genotyping methods have been developed for Campylobacter (Nielsen et al. 2000; Wassenaar and Newell 2000). Multilocus sequence typing (MLST) has been developed and used for the molecular typing Camp. jejuni (Suerbaum et al. 2001; Dingle et al. 2001a,b). These studies have shown that MLST is a useful typing tool for discriminating isolates, defining population structure, identifying trends indicating potential associations among sequence types (STs) or lineages and specific isolation sources or environmental niches and for the identification of possible associations between ST and pathogenicity, despite MLST having a lower discriminatory index than some typing techniques such as pulsed field gel electrophoresis (Sails et al. 2003a,b). The ability to discriminate and group isolates is useful for the identification of potential outbreaks warranting investigation and thus public health intervention.

We describe in this study the genotypic diversity, as defined by MLST type, found among 153 Australian human Camp. jejuni isolates. These isolates represent a subsample of those submitted sequentially to a public laboratory serving a defined geographical region of Australia over a 30-month period.

Methods

Campylobacter jejuni isolates

A case–control study was conducted in the Hunter Health Area of New South Wales, Australia, between January 1999 and July 2001. Two pathology laboratories supplied reports on subjects with Campylobacter detected in stool samples. Of the 318 cases that were eligible and participated, a total of 240 Campylobacter patients were identified through one laboratory. The laboratory had aimed to store as many isolates as possible, of which 171 isolates were stored and remained viable. Of the 171 stored isolates, 153 isolates were Camp. jejuni, as determined by PCR using the method of Linton et al. (1997), and these were genotyped by MLST.

Culture of isolates and preparation of chromosomal DNA

The initial isolation and identification of isolates was performed, as described previously (Sharma et al. 2003). On receipt in our laboratory, the isolates were subcultured onto Columbia agar plates supplemented with 5% horse blood (Oxoid, Adelaide, Australia), and incubated for 48 h at 37 °C under micro-aerophilic conditions using CO2-generating packs (Anaerocult C; Merck) in an anaerobic jar (Oxoid, Basingstoke, UK).

Genomic DNA was prepared essentially by the method of Pitcher et al. (1989) with the following modifications: The harvested bacterial cells were resuspended in lysozyme (10 mg ml−1; Roche Diagnostics GmbH, Mannhein Germany) in TE buffer pH 8 [10 mmol l−1 Tris (Tris (hydroxymethyl)aminomethane), 1 mmol l−1 EDTA (ethylenediaminetetra-acetic acid)] and incubated for 30 min at room temperature. Cell lysis was then performed by the addition of 5 mol l−1 guanidine thiocyanate and 100 mmol l−1 EDTA. DNA was purified by chloroform/phenol/isoamyl alcohol (25 : 24 : 1) extraction and isopropanol (100% v/v) precipitation. The DNA was removed by spooling with a pipette tip, and then transferred into a clean Eppendorf tube containing 80% (v/v) ethanol. The DNA was washed twice in 80% (v/v) ethanol without centrifugation (each wash being removed by aspiration), dried under vacuum and dissolved in TE overnight at 4 °C. The integrity of the DNA was checked by horizontal gel electrophoresis in TAE (40 mmol l−1 Tris, 20 mmol l−1 acetic acid, 2 mmol l−1 EDTA) buffer.

PCR amplification and nucleotide sequence determination

The PCR amplification and nucleotide sequencing primers were used for the seven loci as described previously (Dingle et al. 2001a) with the following modifications. During PCR amplification, the time for denaturation at 94 °C was reduced to 1 min, while the number of cycles was increased to 40. The amplification products were purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The nucleotide sequences were determined using the BigDye Terminator v2 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Unincorporated dye was removed with 75% isopropanol as per the manufacturer's instructions, and the reaction products were detected with the ABI Prism 3700 Capillary Sequencer (Applied Biosystems). Sequences were aligned and assembled using the GeneBase computer program (Applied Maths, Kotrijk, Belgium).

Assignment of allele numbers, sequence types and clonal complexes

Allele numbers, STs and clonal complex assignments were obtained by interrogation of the Campylobacter MLST database (http://pubmlst.org/campylobacter/). Previously unidentified allele sequences and STs were submitted to the database for the assignment of the next number in sequence and these were added to the database.

Laboratory of enteric pathogens serotyping

This was performed according to the method described by Frost et al. (1998) with modification. This direct agglutination method employed absorbed antisera. Antisera were obtained from the Campylobacter Reference Unit, LEP, Central Public Health Laboratory, Colindale, UK. Each isolate was first tested with 12 antisera raised against ‘common’ serotypes. Only strains that did not react with these initial antisera were then tested with the full panel of 65 antisera. Reference antisera were diluted 40-fold and 25 μl aliquots were dispensed into U-bottom microtitre trays. Direct agglutination was performed using growth harvested from 24- to 48-h plate culture, suspended in 1 ml of phosphate-buffered saline (PBS) to produce a dense suspension that was heated at 100 °C for 30 min. The boiled suspension was added to 5 ml PBS to achieve opacity equivalent to McFarland Standard 4. Aliquots of 25 μl of the test suspension were added to the microtitre trays containing antisera. The trays were incubated at 50 °C in a moist atmosphere with gentle agitation in an orbital shaker and agglutination was read after 2 h. Isolates that did not agglutinate were incubated for a further 30 min and read again. Further titration of isolates agglutinating more than one standard antiserum was not undertaken. Thus, results were reported as all serotypes to which agglutination was detected, e.g. 13/30/52 would be the result for an isolate that agglutinated antisera to serotypes 13, 30 and 52.

Results

Isolate representativeness

The isolates included in this study comprised those stored at the public laboratory of the Hunter Health Area, and services a different population from many of the private laboratories. Of the 240 cases identified through the public laboratory, 90 (37·5%) were admitted to hospital compared with 3 (3%) hospitalized cases of the 115 identified through the other pathology service provider (Yates corrected χ2 = 47·2, P < 0·001). Thus, cases of Camp. jejuni included in this study were likely representative of more severe cases.

Diversity of sequence types

A total of 40 STs were identified among the 153 isolates (Table 1). Of these, 19 STs (66 isolates, 43%) were newly described and were submitted to the database, while the remaining 21 STs (87 isolates, 57%) had been previously isolated in other geographical locations. Three STs, ST-48, ST-257 and ST-528, were the predominant STs isolated and accounted for nearly 40% (59/153) of all the isolates. Nineteen STs occurred only once in the data set, with ten of these being newly described. Of the 19 new STs, nine were represented by more than one isolate, with ST-523, ST-525, ST-528, ST-530 and ST-531 accounting for 73% (48/66) of these.

Table 1.   Clonal complexes and sequence types (ST) of Campylobacter jejuni
Clonal complexSTNo. of isolatesAllelic profile of STLaboratory of enteric pathogen serotypes
aspAglnAgltAglyApgmtktuncA
  1. New allele numbers and sequence types shown in bold face. UT, untypeable; 11/13, isolate that agglutinated serotypes 11 and 13 antisera.

48482324127154, 50, UT
257197275246245611, UT
257189246245611, 31, 11/13/31, 14/50, UT
5322924621055611/13, UT
3543543810221112618
528187410221112618
212122113215UT
507211232151, UT
53121213215UT
19012153235UT
45122123235UT
5361217123215UT
569128053235UT
52525925210223613, 31, 11/13, 11/13/31, UT
701925210953631
16141421210863631, 11/31, 13/31, 11/13/31
53319172948636UT
35353725210363, 60, UT
524281752103137, UT
527277752103618, 60
53712175211816UT
6583121144524193618
523724193113618, 50, UT
4525247101171UT
4514710417127
233127104171UT
529147910442511UT
5381471044225121
607525672510113715, 50, 5/50/60/62, 50/60/62
206227524522154, 13, UT
42424123459323, UT
616111422631750
56811464263179
225671136433119
4435117172152331237
4605351930228959613
Unassigned44917715621167663
531102715621167614, 18, 50, 14/50
526121542713802314
5307922108659512, 23, 12/37, UT

The new STs resulted from either new allele sequences (n = 11) or resulted from new combinations of previously described alleles (n = 8; Table 1). New allele sequences were found for aspA (n = 2), glnA (n = 3), gltA (n = 1), glyA (n = 2), pgm (n = 1) and tkt (n = 2). No new allele sequences were found for uncA.

Clonal complexes

Thirty-six STs, representing 134 (88·2%) isolates were assigned to 15 previously described clonal complexes or lineages, with an identifiable ancestral type (Table 1). Two of the remaining four STs (ST-526 and ST-530), both of which were new, could not be assigned to any of the known lineages. The remaining two unassigned STs, ST-449 and ST-531, formed a cluster of two differing by only a single nucleotide in the aspA allele, suggesting clonal relatedness. Similarly, the new STs, ST-197, ST-532 (both assigned to the ST-257 complex), ST-528 (ST-354 complex) and ST-568 (ST-61 complex) differed from their respective ancestral type by only a single nucleotide in a single allele vis-à-vis ST-197 and ST-257, aspA; ST-532 and ST-257, pgm; ST-528 and ST-354, aspA; and ST-568 and ST-61; gltA. Of the 15 clonal complexes identified, eight (ST-21, ST-45, ST-52, ST-61, ST-257, ST-353, ST-354 and ST-658) were represented by multiple STs. ST-21 complex was represented by the largest number of different STs (n = 7), including two new STs, ST-536 and ST-569. ST-48 complex was the most frequently represented with 23 isolates, all of which were ST-48, the ancestral type (Fig. 1). ST-257 complex was the second most frequently represented, with 22 isolates, the majority of which (18/22) were also the ancestral type, ST-257. In contrast to the ST-48 and ST-257 complexes, the ST-354 complex, which was the third most frequently represented complex, was represented primarily by ST-528 (18/21), a new ST. Four clonal complexes, ST-21, ST-45, ST-257 and ST-353, each had multiple new members identified.

Figure 1.

 Distribution of clonal complexes of 153 Campylobacter jejuni isolates from Australia, 1999–2001.

Temporal distribution

The temporal distribution of the isolates is shown in Fig. 2. Of the major STs (number of isolates ≥7), ST-48, ST-50, ST-257, ST-523, ST-530 and ST-531 were found throughout the study period, suggesting that these STs were endemic in the Hunter region. In contrast, ST-528 was only first isolated in June 2000, nearly 18 months in the study but was then the most frequently isolated ST for the remainder of the study.

Figure 2.

 Temporal distribution of major sequence types of 153 Campylobacter jejuni isolates from Australia, 1999–2001. (bsl00022) ST-48, (bsl00021) ST-50, (bsl00020) ST-257, (bsl00018) ST-523, (bsl00023) ST-528, (bsl00068) ST-530, (bsl00004) ST-531 and (bsl00001) other.

Laboratory of enteric pathogen serotypes

A large proportion of isolates were non-typeable using this method (51/153, 33%). Among the typeable isolates, different laboratory of enteric pathogen (LEP) serotypes were found in association with the same ST (e.g. ST-257 and ST-525). Greater LEP serotype variability was generally observed among the STs identified in greater numbers. One exception was ST-528, all 18 isolates of which were LEP serotype 18 as was ST-354, a member of the same clonal complex as ST-528. This LEP serotype was also expressed by one ST-312 isolate, one ST-523 isolate, one ST-527 isolate and four ST-531 isolates.

Overseas travel

Seven isolates were collected from patients experiencing symptoms within 7 days of return from overseas travel, suggesting probable acquisition outside Australia. All seven isolates were different STs; ST-70, ST-161, ST-451, ST-525, ST-529, ST-530 and ST-533 (Table 2). Of these STs, four (ST-525, ST-529, ST-530 and ST-533) were new to the database. Four STs (ST-161, ST-451, ST-525 and ST-530) were also isolated from patients who did not travel overseas.

Table 2.   Sequence types (STs) of travel acquired Campylobacter jejuni
STClonal complexTravelled toSpecimen dateLaboratory of enteric pathogen serotype
  1. UT, untypeable; NA, not assigned.

  2. †Only found among cases that traveled.

70† 52Hong KongSeptember 200031
161 52ThailandFebruary 199913, 31
451 21New ZealandOctober 2000UT
525607Indonesia (Bali)July 199950, 60, 62
529† 45FijiJanuary 2001UT
530NAIndonesia (Bali)April 200112, 37
533† 52IndonesiaJuly 2001UT

Discussion

As part of an Australia-wide multicentre typing project investigating different subtyping methods, we have used MLST to examine the diversity and population structure of Camp. jejuni isolates associated with gastrointestinal infections in humans. The Camp. jejuni isolates, representing those submitted to a public laboratory from sporadic cases, were collected during the 30-month period from January 1999 to July 2001 in the Hunter region of New South Wales as part of a longitudinal case–control study. MLST has been demonstrated to effectively discriminate between Camp. jejuni isolates from a wide range of sources and geographical locations as well as to allow the study of the population structure and the evolutionary mechanisms of this organism (Dingle et al. 2001a; Suerbaum et al. 2001; Duim et al. 2003; Sails et al. 2003a,b). This is the first study of the use of MLST to examine Camp. jejuni isolates collected sequentially over an extended time period from a discrete geographical region in the Southern Hemisphere.

Consistent with the findings of Duim et al. (2003) who also examined sequentially collected Camp. jejuni isolates over a 12-month period in Curacao, the Camp. jejuni isolates from the Hunter region were found to be highly diverse, with a total of 40 different STs identified. Of these, 19 were new to the database, while the remaining 21 had been previously described in other geographical locations. At the time of preparation, only one of the new STs, ST-538, had been isolated outside of this collection. These findings are consistent with those of Duim et al. (2003) who also identified STs that had been previously described as well as those only identified from, and possibly unique to, Curacao. The high level of diversity and low frequency of numerous STs, 19 of which were only detected once, confirm the sporadic nature of Campylobacter infections. Although a diverse range of STs were found, nearly half (45%) of the isolates were represented by only four STs, ST-48, ST-257, ST-528 and ST-531 (n ≥ 10). These findings are similar to those in Curacao where only a limited number of STs, albeit different to those in our data set, also accounted for the majority of isolates (Duim et al. 2003). ST-48 and ST-257 are also commonly found in the United Kingdom (Dingle et al. 2002), while ST-48 has also been found in Japan, Curacao, the Netherlands, Scotland and the United States (http://pubmlst.org/campylobacter/). These two STs have been isolated from a number of sources, including human infections, beef, lamb and poultry. In contrast, ST-528 and ST-531 have not, as yet, been isolated outside of Australia and have so far only been isolated from the cases of human infection. This may suggest that certain STs have greater pathogenic potential than others, and thus become established as the predominant cause of disease.

Seven isolates were collected from patients within 7 days of overseas travel suggesting overseas acquisition. All seven isolates were of different STs, with four of these STs also isolated from patients who did not travel overseas. Of the remaining three STs, two were new to the database, while the third ST (ST-70) had been previously described in association with human infection.

These findings may suggest that some STs have a global distribution, while others may be more restricted in their distribution, possibly to a local environment. However, further investigation would be necessary to test this hypothesis.

Of the 19 new STs, 11 contained new allele sequences. Of these, ten differed from known alleles by a single nucleotide, suggestive of either point mutations or recombination events, while the remaining new sequence differed at multiple sites, suggestive of recombination events (Sails et al. 2003a,b). The remaining eight new STs resulted from new combinations of previously identified allele sequences, suggestive of recombination events.

The majority (36/40; 90%) of STs identified in this study were assigned to a previously described clonal complex compared with 92% described by Dingle et al. (2002) and 93% by Duim et al. (2003). The ST-21 clonal complex was represented by the largest number of different STs (n = 7). This finding is consistent with those of Duim et al. (2003) in Curacao and data from the PubMLST database. In addition, consistent with Duim et al. (2003), but in contrast to the PubMLST database in which the ST-21 complex is also represented by the largest number of isolates, the ST-21 clonal complex was not represented by the most isolates. In our data set, the largest number of isolates was from the ST-48 clonal complex; all of which were ST-48, the ancestral type, suggesting that this ST may be well adapted to this region.

Examination of temporal trends for these STs showed that ST-48, ST-257 and ST-531 were found throughout the study period, suggesting that these types are endemic in the population (Fig. 2). National monthly notifications for Campylobacter during the study period did not display any marked seasonal trend; however, a small summer peak was observed in summer 2000–2001(Communicable Diseases Network Australia). Isolates comprising the collection approximately mirrored the frequency of notifications with the exception of February and June 2001, when greater numbers of isolates were collected compared with other months. Using pulsed field gel electrophoresis, Hanninen et al. (2000) also demonstrated both the persistence and diversity of Camp. jejuni genotypes in the Helsinki area over a 3-year period. Two New Zealand studies of PFGE-Penner serotyping have also described temporal trends in Campylobacter infections (Hudson et al. 1999; Gilpin et al. 2006). In contrast to ST-48, ST-257 and ST-531, ST-528 was first isolated in June 2000, nearly 18 months in the study. It then underwent a rapid increase in incidence, becoming the most frequently isolated single ST for the remainder of the study period. Further examination would be necessary to determine if this ST has become established in the Hunter region or may have represented a localized cluster. ST-528 was new to the database, and at the time of preparation (November 2005), the only representation of this ST in the database remains from this study. ST-528 is a member of the ST-354 clonal complex, and differs from ST-354 (the ancestral founding member) by a single nucleotide change in the aspA allele, which resulted in a new sequence. Of interest, ST-354 was isolated only during 1999 (n = 3) and not after the appearance of ST-528. This suggests the possible evolution of ST-354–ST-528 or its replacement in a particular niche by ST-528 and the concomitant emergence of ST-528 in this community. However, as the data set is relatively small and the time interval of study is short, it would be difficult to draw any firm conclusions from this observation.

Examination of LEP serotyping in comparison to MLST was undertaken. There were 15 clonal complexes detected in this study, with one or more serotypes identified among the isolates for each clonal complex (Table 1). Of the 15 clonal complexes, 11 (ST-21, ST-22, ST-42, ST-45, ST-48, ST-52, ST-61, ST-206, ST-257, ST-353 and ST-354) are described by Dingle et al. (2002), with resultant serotypes. Serotypes found by Dingle et al. (2002) associated with those 11 clonal complexes were also found among isolates from 10 clonal complexes in this study (ST-21, ST-22, ST-42, ST-45, ST-48, ST- 52, ST-61, ST-206, ST-257 and ST-353). The remaining clonal complex, ST-354, contained serotypes not previously described by Dingle et al. (2002) but are described on the PubMLST database. Serotypes were found in association with six clonal complexes vis-à-vis: ST-52 (serotype 13), ST-61 (serotype 9), ST-257 (serotype 14/50), ST-353 (serotypes 18 and 60), ST-607 (all serotypes) and ST-658 (all serotypes), which have not been described previously for these complexes. The remaining clonal complexes, ST-443 and ST-460, contained serotypes previously described in the PubMLST database. While serotypes were identified among isolates from all of the clonal complexes identified in this study, the limitations of serotyping were reinforced by the fact that one third of all isolates (51/153) were not typed by this method. The largest number of untypeable isolates were from the ST-21 clonal complex, with only two of the ST-50 isolates being typed as serotype 1. The remaining ST-50 isolates and all isolates from the six other STs in this complex were untypeable. Two other STs, ST-48 and ST-257, also had high proportions of isolates that were untypeable, 8/23 (35%) and 9/18 (50%), respectively. Serotypes 4, 11, 13, 18, and 50 were detected among isolates from more than one clonal complex. These data support the antigenic variability of Campylobacter.

In this study, we have only examined Camp. jejuni isolates associated with human disease and not from other sources known to be risk factors for the transmission of infection. Meat, especially chicken, is the most common vehicle of Campylobacter infection. The majority of chicken is produced within the country with very limited import of cooked chicken meat. Thus, import of meat is unlikely to be responsible for the presence of STs that appear to have a global distribution, nor for the diversity of STs seen in the Australian population. The large proportion of Australian isolates comprising STs unique to Australia suggests a local source of disease. Further investigations of Campylobacter from known sources will be necessary to examine the links with human disease.

While MLST is well established as a tool for examining the population structure of bacterial populations, the ability of MLST to discriminate between subtypes and to assign an isolate to a clonal complex may be useful in providing a guide to the potential source of an isolate, and thus be a useful tool in the epidemiological investigation of Campylobacter infections. The value of this may be seen in the findings of Dingle et al. (2002) who identified possible associations between certain clonal complexes and the source of an isolate. For example, the ST-45 and ST-257 clonal complexes consisted of isolates predominantly from human disease and chickens, while the ST-42 and ST-61 complexes had isolates predominantly from human disease, cattle and sheep.

This study has highlighted the value of using a typing method with access to a centralized database. By utilizing the existing Campylobacter MLST database (http://pubmlst.org/campylobacter/), we have been able to compare directly isolates from the Hunter region of New South Wales with isolates from around the world. This has enabled us to identify STs in Australia that have a global distribution, as well as to identify STs that appear, at least currently, to be unique to our continent. Thus, the examination of Camp. jejuni from a specific geographical location, such as Australia, provides further insight into the epidemiology and population structure of Campylobacter. The use of a centralized database also allowed the deposition of both new sequences and allelic profiles, which will continue to build the database and further the understanding and epidemiological study of Camp. jejuni worldwide. These results highlight the genetic diversity of Camp. jejuni isolates associated with human gastroenteritis in the Hunter region of New South Wales. The high incidence of new STs, as well as the high frequency of a limited number of possibly endemic STs, demonstrates the usefulness of MLST as an epidemiological tool for the investigation of Camp. jejuni infections. These findings, combined with the fact that the majority of isolates submitted to the Camp. jejuni database have been derived from the Northern Hemisphere, suggest that further investigation of Camp. jejuni isolates from around Australia would be invaluable in the epidemiological study of this organism, both in this country and in a global setting.

Acknowledgements

This Study was funded, in part, by the OzFoodNet program of work, which is an initiative of the Australian Government Department of Health and Ageing. This publication made use of the Campylobacter jejuni Multi Locus Sequence Typing website (http://pubmlst.org/campylobacter/) developed by Keith Jolley and Man-Suen Chan and sited at the University of Oxford (Jolley et al. (2004) BMC Bioinformatics, 5, 86). The development of this site was funded by the Wellcome Trust and continued support is currently provided by the UK Department for Environment, Food and Rural Affairs.

Australian Campylobacter Subtyping Study Group: (listed in alphabetical order): Penny Adamson (Flinders Medical Centre, South Australia), Kellie Cheung (Institute of Clinical Pathology and Medical Research, Westmead, New South Wales), Barry Combs (OzFoodNet, Department of Human Services, Adelaide, South Australia), Craig Dalton (Hunter New England Population Health, Newcastle, New South Wales), Steve Djordjevic (Elizabeth Macarthur Agricultural Institute, Camden, New South Wales), Robyn Doyle (Institute of Medical and Veterinary Science, Adelaide, South Australia), John Ferguson (Hunter New England Health Service, Newcastle, New South Wales), Lyn Gilbert (Institute of Clinical Pathology and Medical Research, Westmead, New South Wales), Rod Givney (Department of Human Services, Adelaide, South Australia), David Gordon (Flinders Medical Centre, Bedford Park, South Australia), Joy Gregory (OzFoodNet, Department of Human Services, Melbourne, Victoria), Geoff Hogg (Microbiological Diagnostic Unit, University of Melbourne, Parkville, Victoria), Tim Inglis (Division of Microbiology and Infectious Diseases, PathWest, Nedlands, Western Australia), Peter Jelfs (Institute of Clinical Pathology and Medical Research, Westmead, New South Wales), Martyn Kirk (OzFoodNet, Canberra, Australian Capital Territory), Karin Lalor (OzFoodNet, Department of Human Services, Melbourne, Victoria), Jan Lanser (Institute of Clinical Pathology and Medical Research, Westmead, New South Wales), Lance Mickan (Institute of Medical and Veterinary Science, Adelaide, South Australia), Lyn O'Reilly (Division of Microbiology and Infectious Diseases, PathWest, Nedlands, Western Australia), Rosa Rios (Microbiological Diagnostic Unit, Parkville, Victoria), Minda Sarna (OzFoodNet, Department of Health, Perth, Western Australia), Hemant Sharma (Hunter New England Health Service, Newcastle New South Wales), Helen Smith (Queensland Health Scientific Services, Coopers Plains, Queensland), Russell Stafford (OzFoodNet, Queensland Health, Queensland), Leanne Unicomb (OzFoodNet, Hunter New England Population Health and National Centre for Epidemiology and Population Health, Australian National University, Canberra, ACT), Mary Valcanis (Microbiological Diagnostic Unit, University of Melbourne, Parkville, Victoria).

Ancillary