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Systematic and Applied Microbiology 33 (2010) 7–14

Contents lists available at ScienceDirect

Systematic and Applied Microbiology

0723-20

doi:10.1

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journal homepage: www.elsevier.de/syapm

Reassessment of the taxonomy of Arcobacter cryaerophilus$

Lies Debruyne a, Kurt Houf b, Laid Douidah b, Sarah De Smet b, Peter Vandamme a,n

a Laboratory of Microbiology (WE 10), Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgiumb Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Belgium

a r t i c l e i n f o

Keywords:

Arcobacter

Arcobacter cryaerophilus

Intraspecies diversity

Taxonomy

Polyphasic taxonomy

AFLP analysis

hsp60 gene sequence analysis

20/$ - see front matter & 2009 Elsevier Gmb

016/j.syapm.2009.10.001

e: Nucleotide sequence data reported are a

k databases under the accession num

59–FN555367, FN555369–FN555375, and FN

esponding author.

ail address: Peter.Vandamme@UGent.be (P. V

a b s t r a c t

Arcobacter cryaerophilus is a heterogeneous species in which two distinct subgroups have been reported.

In the present study, the taxonomic status of these subgroups was reassessed using amplified fragment

length polymorphism and heat shock protein 60 gene sequence analysis. The results demonstrated that

A. cryaerophilus has a complex taxonomic structure, which consists of multiple cores of strains that

share intermediate levels of DNA–DNA hybridisation and exhibit low levels of DNA–DNA hybridisation

towards other Arcobacter species. One of these cores consisted of the majority of strains and included

most subgroup 2 strains from previous studies. A. cryaerophilus subgroup 1 strains represented three

distinct cores, among which the type strain occupied a distinct position. These results therefore also

demonstrate that the current subgroup nomenclature in A. cryaerophilus should be abandoned, and that

the type strain is genetically aberrant and poorly represents strains belonging to this species.

& 2009 Elsevier GmbH. All rights reserved.

Introduction

In recent years, there has been an increased interest inthe genus Arcobacter for its potential role as an emerginghuman pathogen. The genus belongs to the family of Campylobac-

teraceae, and currently comprises 8 species. Arcobacter butzleri,A. cryaerophilus, Arcobacter skirrowii, Arcobacter cibarius and Arco-

bacter thereius have been isolated from a wide range offarm animals, their meat and water sources; the formerthree species have also been associated with human illness[1,3,15–17,29,30,33,37,40,42,46]. Arcobacter halophilus (isolatedfrom a hypersaline lagoon) [7], Arcobacter nitrofigilis (isolated fromSpartina alterniflora roots) [26], Arcobacter mytili (isolated from amarine environment) [4], and a candidate species, ‘Candidatus

Arcobacter sulfidicus’, a highly motile sulphide-oxidizing bacterium[45], are all free-living, environmental bacteria.

From its first description [28], it was clear that A. cryaerophilus

represented a heterogeneous taxon. This initial observation wasbased on a wide range of biochemical and physiological tests, andwas later supported by chemotaxonomic and genotypic data[14,24,41]. Based on the observations of Kiehlbauch et al. [24] andVandamme et al. [41], two subgroups referred to as subgroups 1Aand 1B [24], or 1 and 2 [41] were distinguished. The aim of thepresent study was to reassess the taxonomy of A. cryaerophilus,

H. All rights reserved.

vailable in the DDBJ/EMBL/

bers FN257283–FN257300,

555377–FN555392.

andamme).

using amplified fragment length polymorphism (AFLP) and hsp60

gene sequence analyses. AFLP analysis combines accurate species-level identification with the capacity to reveal intraspeciesrelationships, which is particularly interesting in heterogeneousspecies like A. cryaerophilus. It was first described by Vos et al. [44]as a fingerprinting technique for samples of any origin orcomplexity, and the usability of this technique as a tool inbacterial taxonomy was soon afterwards evaluated by Janssenet al. [19]. Since then AFLP analysis has been applied to a widevariety of genera, for identification and typing purposes[21,23,27,29,35,36]. In the present study, the AFLP protocoldescribed by Duim et al. [10], which proved very useful to studythe diversity in the closely related genus Campylobacter [6,8,9],was optimized to study the genus Arcobacter, with a particularemphasis on A. cryaerophilus.

The hsp60 gene encodes the 60 kDa chaperonin protein that isfound in virtually all bacteria, and the utility of this targetfor phylogenetic analysis is well established [2,11,13,25,43].Karenlampi et al. [22] introduced phylogenetic analysis basedon the hsp60 gene for the genus Campylobacter; Hill et al. [12]used this approach to separate Campylobacter from the closelyrelated genera Arcobacter and Helicobacter[12].

Materials and methods

Bacterial strains and growth conditions

A total of 101 Arcobacter strains were analyzed in the presentstudy (Table 1). A. butzleri (n=15), A. skirrowii (n=10), A. thereius

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Table 1Strain collection. LMG, BCCM/LMG Bacteria Collection, Ghent University, Ghent,

Belgium (additional information on these strains: www.straininfo.net).

Species Strain no Source (origin,isolation year)

A. butzleri LMG 9910 Deadborn piglet

(UK)

LMG 10828T Human diarrheic

faeces (USA)

LMG 10898, LMG 10900,

LMG 11118, LMG 11119,

LMG 11120, LMG 11124,

LMG 11125

Human faeces (Italy,

1983) - outbreak

LMG 14714 Human faeces

(Greece)

R-325, R-8636 Human faeces

(Belgium)

R-700 (Germany, 1996)

R-14600, R-14606 (Turkey)

A. skirrowii LMG 6621T Ovine diarrheic

faeces (UK, 1980)

LMG 8538 Cow with

abdomastitis (UK,

1979)

LMG 9911 Aborted porcine

fœtus, thoracal fluid

(UK)

LMG 10234 Aborted porcine

fœtus, liver & lung

(Canada)

LMG 14983, LMG 14988 Bull, preputial fluid

(USA)

R-3544, R-3545, R-36432,

R-36844

Aborted porcine

fœtus, liver & kidney

(Denmark, 1996-

1997)

A. cibarius LMG 21996T, LMG 21997,

LMG 21998

Broiler skin

(Belgium, 2002)

R-16578, R-16585 Broiler skin

(Belgium, 2002)

A.cryaerophilus-subgroup 1

LMG 6622a Aborted swine

fœtus, kidney (UK)

LMG 9065a Aborted ovine fœtus,

placenta (UK)

LMG 9863 Aborted ovine fœtus

(UK)

LMG 9865 Aborted porcine

fœtus (UK)

LMG 9871 Aborted bovine

fœtus, kidney (UK)

LMG 10210 Aborted bovine

fœtus (Canada)

LMG 24291T,a Aborted bovine

fœtus, brain (UK)

A.cryaerophilus-subgroup 2

LMG 7537 Aborted ovine fœtus

(UK)

LMG 9861 Aborted bovine

fœtus, peritoneal

fluid (UK)

LMG 9864, LMG 9870 Aborted porcine

fœtus, eye (UK)

LMG 9867 Aborted equine

fœtus, spleen (UK)

LMG 9909b Deadborn piglet

(UK)

LMG 9937, LMG 9944, LMG

9947, LMG 9948

Aborted fœtus

(Canada)

LMG 10209, LMG 10211,

LMG 10212

Aborted bovine

fœtus (Canada)

LMG 10213, LMG 10215 Aborted porcine

fœtus (Canada)

LMG 10216, LMG 10217,

LMG 10218, LMG 10222

Aborted porcine

fœtus, placenta

(Canada)

LMG 10219, LMG 10221,

LMG 10226, LMG 10229,

Table 1. (continued )

LMG10232, LMG 10233, LMG

10235, LMG 10241

Aborted porcine

fœtus, kidney

(Canada)

LMG 10228 Aborted porcine

fœtus, liver, lung &

spleen (Canada)

LMG 10231, LMG 10237,

LMG 10244

Aborted porcine

fœtus, lung & liver

(Canada)

LMG 10230 Porcine diarrheic

faeces (Canada)

LMG 10829b (subgroup 2

reference)

Human blood

(Belgium)

A. cryaerophilus R-326, R-1633, R-1632, R-

2339, R-3135, R-8631

Human faeces

(Belgium)

R-1742, R-12811, R-12813,

R-14234

Human (Belgium)

R-3527, R-3533, R-4016, R-

36434, R-36447, R-36853

Aborted porcine

fœtus, liver & kidney

(Denmark, 1996-

1997)

R-4043, R-4044 Poultry (France)

R-36851 Duck cloacal content

(Denmark)

A. thereius LMG 24486T, LMG 24487 Aborted porcine

fœtus, liver & kidney

(Denmark, 1996-

1997)

R-36843, R-36845, R-36849 Aborted porcine

fœtus, liver & kidney

(Denmark, 1996-

1997)

R-36855, R-36856, R-36857 Duck cloacal content

(Denmark)

A. nitrofigilis LMG 7604T, LMG 7547 Spartina alterniflora,

roots or root-

associated sediment

(USA)

A. halophilus R-31813T (LA31BT) Water from

hypersaline lagoon

(USA, 2000)

A. mytili LMG 24559T Marine environment

(Spain)

R, refers to non-public strain collection.

a Hybridization group 1A.b Hybridization group 1B (Kiehlbauch et al., [24]).

L. Debruyne et al. / Systematic and Applied Microbiology 33 (2010) 7–148

(n=8), A. cibarius (n=5), A. cryaerophilus (n=59), and A. mytili

(n=1) strains were grown on the Mueller–Hinton agar (Oxoid)supplemented with 5% horse blood, while for A. nitrofigilis (n=2)and A. halophilus (n=1) the blood agar was supplemented with 2%NaCl. All strains were incubated micro-aerobically at 28 1C for48–72 h. For A. cryaerophilus, 7 and 33 strains had been previouslyassigned to subgroups 1 or 2, respectively, based on the whole-cell protein SDS-PAGE analysis [41].

AFLP analysis

DNA was extracted according to the method described byPitcher et al. [31]. DNA integrity was checked by agarose gelelectrophoresis. DNA was quantified, and checked for purity byoptical density measurements at 234, 260 and 280 nm (Spec-tramax 384plus spectrophotometer, Molecular Devices). DNAconcentrations were standardized at 250 ng/ml.

For AFLP analysis, a modified version of the AFLP protocoldescribed previously [10] was used. One microgram of chromo-somal DNA was digested with 5U HindIII and 5U HhaI (NewEngland Biolabs) for 30 min, and was subsequently ligated to

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Table 2Primer sequences for AFLP analysis.

Primer name Sequence (5030)

H00 GACTGCGTACCAGCTT

tH00 GATGAGTCCTGATCGC

H01 GACTGCGTACCAGCTTA

tH01 GATGAGTCCTGATCGCA

L. Debruyne et al. / Systematic and Applied Microbiology 33 (2010) 7–14 9

restriction site-specific adapters for 1 h, both at 37 1C. No pre-selective amplification was performed, but the purified DNAtemplates were directly used in a selective PCR reaction. For PCR,1.5 ml of the sample was amplified in a 10 ml (final volume)mixture containing 7.5 ml amplification core mix (ACM) (AppliedBiosystems), 2.5 pmol of HhaI primer and 2 pmol of fluorescentlylabelled (6-FAM) HindIII primer. The following primers weretested: a HindIII and HhaI primer without selective nucleotide,H00-FAM and tH00, respectively, and a HindIII and HhaI primerwith an additional 30 A nucleotide, H01-FAM and tH01, respec-tively (Table 2). PCR was performed according to the AFLPmicrobial fingerprinting protocol (Applied Biosystems). Onemicroliter of the final product was analyzed by capillary gelelectrophoresis using an ABI 3130xl automated DNA sequencer.For every sample, 0.4 ml of an internal lane standard (LIZ-600,Applied Biosystems) was included.

AFLP data processing

AFLP profiles were collected with the Data Collection softwarev 3.0 (Applied Biosystems). Profiles were imported in BioNu-merics v 4.61 (Applied Maths) for further analysis, using theCrvConv filter. Normalization was performed using the internalLIZ-600 standard. After normalization, the similarity betweenprofiles was determined by the Pearson product momentcorrelation coefficient, expressed as a percentage similarity, anda dendrogram was constructed by numerical analysis of theprofiles, using the UPGMA (unweighted pair group with arith-metic means) algorithm.

hsp60 gene sequence analysis

hsp60 gene sequence analysis was performed as describedpreviously [6,12]. In brief, PCR amplification reactions contained1X PCR buffer, 200 mM dNTP’s, 0.5 U Taq polymerase, 5 pmol ofboth forward primer H729 (50 CGCCAGGGTTTTCCCAGTCACGAC-GAIIIIGCIGGIGAYGGIACIACIA 30) and reverse primer H730(50 AGCGGATAACAATTTCACACAGGAYKIYKITCICCRAAICCIGGIG-CYTT 30), and 50 ng of genomic DNA, with the final volumeadjusted to 50 ml. The amplification primers include landing sitesfor the sequencing primers (underlined), enabling direct sequen-cing [12]. Thermal cycling conditions were as follows: initialdenaturation at 95 1C for 2 min, followed by 30 cycles ofdenaturation at 95 1C for 30 s, annealing at 46 1C for 30 s, andelongation at 72 1C for 30 s, and a final elongation step at 72 1C for5 min. PCR products were purified using the QIAQuick PCRpurification kit (Qiagen). Sequencing reactions were performedusing the BigDyes Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems, Foster City, CA, USA) and purified using the BigDyesXTerminatorTM Purification Kit (Applied Biosystems, FosterCity, CA, USA). Sequences were generated using an AppliedBiosystems 3130xl Genetic Analyzer (Applied Biosystems, FosterCity, CA, USA).

Sequence assembly and alignments were performed usingthe BioNumerics v5.1 software. Ambiguous bases were discarded

for the analysis and a rooted phylogenetic tree was constructedusing the neighbour-joining method (bootstrap analysis, 500replicates), and a maximum-likelihood method.

Results

AFLP analysis

Initial testing revealed that if AFLP profiling was executed withthe H01-FAM/tH01 primer combination [10], the number of peaksobserved in the AFLP patterns was rather low (between 10 and 30peaks in the 20–600 bp range), resulting in an inadequate speciesdiscrimination. Vos et al. [44] reported that the addition of anextra selective nucleotide generally results in a fingerprint, whichis a subset of the original fingerprint, indicating that the use ofselective nucleotides is an accurate and efficient way to select aspecific set of restriction fragments for amplification [44]. So, byreducing the number of selective nucleotides from two in theoriginal protocol, to one or zero, the number of fragments shouldincrease for all included species. For this purpose, a subset of 25strains was analyzed using three additional primer combinations,H00-FAM/tH00, H00-FAM/tH01, and H01-FAM/tH00, and UPGMAdendrograms were generated for AFLP profiles based on everyprimer combination (data not shown) to determine the optimalprimer combination for the genus Arcobacter. Taking into accountthe number of fluorescently labelled fragments and the require-ment for evenly distributed fragments, H00-FAM/tH01 was thepreferred primer combination. With this combination we ob-served a clear separation of different taxa, and 15–60 evenlydistributed fragments were generated in the 20–600 bp sizerange.

Reproducibility levels were evaluated by duplicate analysis of10 strains and were on average 93.3%, which were comparable tothose observed in other AFLP studies. Inter-experiment reprodu-cibility was evaluated by including the same control strain inevery run.

After determination of the optimal primer combination, all 101Arcobacter strains were analyzed by AFLP, with the selectedprimer combination. Profiles were generated for all strains, andpeaks generated in the 20–600 bp range were included foranalysis. Between different species, there were differences inthe number of peaks generated, ranging from approx. 10 to 15 forA. thereius, to 60 peaks and more for A. cryaerophilus.

After numerical analysis, A. butzleri and A. skirrowii formeddistinct clusters at the 20% similarity level, while the A. cibarius

strains formed a far more coherent cluster at a 72% similarity level(Fig. 1). For all these species a comparable number of peaks wereobserved in the AFLP profiles, ranging between 35 and 60 (themajority between 45 and 55). In the A. butzleri cluster, 5 isolatesobtained during an outbreak in Italy [40] formed a tight cluster, ata 97% similarity level, confirming their clonal relationship. Twoother isolates obtained during the same outbreak grouped withthis cluster at a lower similarity level (79%), but visualcomparison revealed that the profiles were near to identical,and differed mainly in peak intensity.

The high level of similarity observed among A. cibarius strains,as compared to A. butzleri and A. skirrowii can be possiblyexplained by the origin of the strains that were included. For A.

cibarius, all strains were isolated from broiler skin during a singlesampling campaign in February 2002 in Flanders (Belgium) [14].For A. butzleri and A. skirrowii, the strain set was far more diverse,with strains varying widely in date of isolation, geographicalorigin and isolation source (Table 1).

For A. cryaerophilus, the majority of strains (n=56) formed asingle cluster above a similarity level of 12% (Fig. 1). This large

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Fig. 1. Dendrogram of AFLP profiles of all Arcobacter species. Similarity was determined by the Pearson product moment correlation coefficient and clustering was performed

by the UPGMA. Strains for which the hsp60 gene sequence was available from public databases or determined in the present study are indicated with an asterisk (n).

L. Debruyne et al. / Systematic and Applied Microbiology 33 (2010) 7–1410

cluster could be separated into several subclusters when applyingthe 20% similarity level observed in A. butzleri and A. skirrowii. Alarge core of 47 strains (subcluster A), two smaller groups

(subclusters B and C), and two separate strains, LMG 10209(belonging to A. cryaerophilus subgroup 2) and R-3533(unassigned), could be differentiated at this similarity level.

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L. Debruyne et al. / Systematic and Applied Microbiology 33 (2010) 7–14 11

Subcluster A included LMG 10829, the strain proposed torepresent the subgroup 2 taxon [24], 29 additional subgroup 2strains [41] and 17 unassigned strains; subcluster B consisted offour subgroup 1 strains [41]; and subcluster C contained the typestrain of A. cryaerophilus (LMG 24291T, subgroup 1) and twosubgroup 2 strains (LMG 9944 and LMG 10216). In addition, threeA. cryaerophilus subgroup 1 strains formed a separate cluster(subcluster D) altogether (Fig. 1).

For A. thereius, strains were separated into two distinctclusters, both at a 50% similarity level, but the number of peaksobserved in the profiles for these strains (ranging between 10 and15), was remarkably lower than for the other Arcobacter species.The two A. nitrofigilis strains formed a tight cluster at a 93%similarity level, and finally, A. halophilus and A. mytili type strainsgrouped separately from all other species.

Some authors have used AFLP analysis for phylogeneticinferences between closely related species [18,20,34], but forArcobacter, the topology of the AFLP dendrogram is discordantwith the phylogenetic relationships as observed by 16S rRNA gene(Fig. S1) and hsp60 gene (Fig. 2) sequence analysis. The presentedAFLP scheme appears well suited for both identification andtyping purposes, as demonstrated by the results for A. butzleri

outbreak strains. However, although the H00-FAM/tH01 primercombination generally yielded the most informative profiles, it issuboptimal for A. thereius, as relatively few bands are generated,which probably led to the subdivision of strains examined intotwo AFLP clusters.

hsp60 gene sequence analysis

Based on AFLP pattern diversity, a total of 54 strains werefurther investigated by means of hsp60 gene sequence analysis(Fig. 1). Fragments of 555 bp in length were compared toArcobacter hsp60 gene sequences from the GenBank, and aneighbour-joining tree (Fig. 2) was constructed. Interspeciessequence similarities ranged between 81.4% (A. halophilus–A. thereius) and 95.5% (A. cryaerophilus–A. skirrowii), intraspeciessimilarities ranged between 96% and 100%, with the exception ofA. cryaerophilus, for which a wider divergence was observed (seebelow).

The 28 A. cryaerophilus strains examined included strains fromall four AFLP subclusters and the two separate strains and againappeared heterogeneous: a large clade comprising the majority ofstrains (including LMG 10829) and two additional small cladeswere delineated while the type strain LMG 24291T occupied adistinct position (Fig. 1). However, eventhough there wereobvious similarities between the AFLP and hsp60 dendrograms(e.g. AFLP subcluster D strains also form a distinct clade in thehsp60 gene sequence analysis), the distribution of the strains wasnot identical (for instance, strain LMG 9861 belongs to subclusterA in the AFLP analysis, but forms a distinct clade with threesubcluster B strains and strain R-3533 in the hsp60 dendrogram).Also, the intraspecies divergence was high, even within the cladeswith values for the largest clade (including LMG 10829) rangingbetween 94.8% and 100%, hence surpassing the divergenceobserved between some A. cryaerophilus and A. skirrowii strains.For the second (consisting of the AFLP subcluster D strains) andthe third hsp60 clades the sequence similarities ranged between97.8% and 99.1% and between 98.4% and 99.8%, respectively.Finally, the type strain LMG 24291T did not show very highsimilarity levels to any of the other A. cryaerophilus strainsincluded in the present study, with similarity values rangingbetween 91.7% and 96.8%.

For A. thereius, the divergence observed in the AFLP analysiswas again supported by the results of the hsp60 gene sequence

analysis. Nevertheless, all strains formed a single clade (Fig. 1),but a higher sequence divergence (95.9–100%) was observedamong members of this species, compared to other Arcobacter

species (A. butzleri: 98.9–100%; A. skirrowii: 97.3–100% andA. cibarius: 99.5–100%).

Discussion

Organisms now known as A. cryaerophilus were first describedin 1985 by Neill et al. [28] as a group of aerotolerantCampylobacter-like organisms, mainly associated with animalabortions. This first description was based solely on extensivebiochemical and physiological examination, but already revealedthat this novel species was heterogeneous. In 1991, Kiehlbauch etal. [24] performed an extensive phenotypic and DNA–DNAhybridisation (DDH) analysis of 78 aerotolerant ‘campylobacters’,and identified two subgroups within A. cryaerophilus, designatedas DNA hybridisation groups 1A and 1B, which were phenotypi-cally indistinguishable. These observations were substantiated ina subsequent study, where the relationships of 77 aerotolerantstrains, all originally identified as Campylobacter cryaerophila,were investigated by using a polyphasic approach [41]. Numericalanalysis of the whole-cell protein profiles [5,38] of 77 strainsrevealed five clusters, three of which formed coherent DDHgroups exhibiting high DDH values (64–98%) within each groupand low (r32%) DDH values towards representatives of othergroups. These three clusters corresponded with A. nitrofigilis, A.

butzleri and the novel species A. skirrowii. The remaining twoclusters were designated as A. cryaerophilus subgroups 1 and 2and corresponded with A. cryaerophilus hybridisation groups 1Aand B, respectively [24]. Although analysis of whole-cell fattyacids further supported the subdivision of A. cryaerophilus intothese two subgroups, the DDH values revealed a rather hetero-geneous subgroup 1 (four DDH values ranging between 56% and67%; on average 60%) and a fairly homogenous subgroup 2(21 DDH values ranging between 56% and 98%; on average 73%).

In spite of the clear correlation between the level of DDH andpercentage of whole-cell protein profile similarity observed insome Arcobacter species (i.e. A. butzleri, A. skirrowii andA. nitrofigilis), and in many other bacterial genera [32,39],assignment of novel A. cryaerophilus isolates by whole-cell proteinanalysis to subgroups 1 or 2 has been increasingly problematic,due to the heterogeneity of A. cryaerophilus-like strains(Vandamme P., unpublished data). In the present study, neitherthe AFLP cluster analysis nor the hsp60 sequence analysissubstantiated the two A. cryaerophilus subgroups delineated byKielhbauch et al. [24] and Vandamme et al. [41]. Although themajority of the subgroup 2 strains from previous studies (Table 1)formed a coherent AFLP cluster (subcluster A – Fig. 1) and hsp60

sequence clade (Fig. 2) in accordance with their high DDH values,A. cryaerophilus subgroup 1 strains from previous studies groupedin AFLP subclusters B and D, while the type strain occupied adistinct position, grouping rather remotely with two subgroup 2strains in subcluster C (Fig. 1). Interestingly, the type strain andsubgroup 1 strains from subclusters B (LMG 6622) and D (LMG9065) exhibited DDH levels in the range 56–67%, and similar DDHvalues in the range 48–69% were obtained towards subgroup 2strains [41]. Furthermore, the same three subgroup 1 strains (thetype strain and strains LMG 6622 and LMG 9065) each belong to adistinct hsp60 sequence clade (Fig. 2). Although in the practise ofpolyphasic taxonomy, 70% DDH level is not to be considered astrict threshold level for species delineation [39], these valuesreveal a considerable genetic divergence among subgroup 1strains and towards subgroup 2 strains. A genetically moreuniform subgroup 2 taxon and hybridisation levels among

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1%Arcobacter butzleri LMG 14714 (FN555359)Arcobacter butzleri LMG 10828T(Dq059474)Arcobacter butzleri LMG 9910 (FN555360)Arcobacter butzleri LMG 10898 (FN555361)Arcobacter butzleri LMG 11118 (FN555362)Arcobacter butzleri LMG 6620 (AY628390)

Arcobacter thereius LMG 24487 (FN257293)Arcobacter thereius R-36843 (FN555363)Arcobacter thereius R-36849 (FN555364)Arcobacter thereius R-36845 (FN257294)Arcobacter thereius R-36856 (FN555365)Arcobacter thereius R-36855 (FN257295)Arcobacter thereius R-36857 (FN555366)Arcobacter thereius LMG 24486T (FN257292)Arcobacter cibarius LMG 21996T (FN257296)Arcobacter cibarius LMG 21997 (FN555367)Arcobacter cibarius LMG 21998 (FN257297)Arcobacter cibarius R-16578 (FN555369)

Arcobacter cryaerophilus LMG 24291T (FN257285)Arcobacter cryaerophilus LMG 9871 (FN555370)Arcobacter cryaerophilus LMG 9863 (FN555371)Arcobacter cryaerophilus LMG 9065 (FN257286)

Arcobacter cryaerophilus LMG 6622 (FN257284)Arcobacter cryaerophilus LMG 10210 (FN555372)Arcobacter cryaerophilus LMG 9861 (FN555373)Arcobacter cryaerophilus LMG 9865 (FN555374)Arcobacter cryaerophilus R-3533 (FN257283)

Arcobacter skirrowii LMG 10234 (FN257300)Arcobacter skirrowii R-36432 (FN555375)

Arcobacter skirrowii R-3544 (FN555377)Arcobacter skirrowii LMG 6621T (DQ059471)Arcobacter skirrowii LMG 8538 (FN555378)Arcobacter skirrowii LMG 14988 (FN555379)Arcobacter skirrowii R-36844 (FN555380)

Arcobacter cryaerophilus LMG 9870 (FN555381)Arcobacter cryaerophilus R-1742 (FN555382)

Arcobacter cryaerophilus LMG 9864 (FN555383)Arcobacter cryaerophilus LMG 9909 (FN257291)Arcobacter cryaerophilus LMG 9867 (FN555384)

Arcobacter cryaerophilus LMG 10222 (FN555385)Arcobacter cryaerophilus LMG 9937 (FN555386)

Arcobacter cryaerophilus R-3527 (FN555387)Arcobacter cryaerophilus LMG 10209 (FN257289)Arcobacter cryaerophilus R-4044 (FN555388)

Arcobacter cryaerophilus LMG 10235 (FN257288)Arcobacter cryaerophilus R-14234 (FN555389)Arcobacter cryaerophilus R-12813 (FN555390)

Arcobacter cryaerophilus LMG 7537 (FN257287)Arcobacter cryaerophilus LMG 10229 (FN555392)Arcobacter cryaerophilus LMG 10829 (DQ059481)Arcobacter cryaerophilus R-326 (FN555391)

Arcobacter nitrofigilis LMG 7604T (DQ059460)Arcobacter halophilus LA31BT (FN257298)

Arcobacter mytili LMG 24559T (FN257299)

8962

57

100

100100

979797

100

100

94

86

100

9799

62

605484

97

100

70

100

67100

8862

100

73

87

99

100

77100

Fig. 2. Rooted neighbour-joining tree based on partial hsp60 gene sequences. All sequences are 555 bp in length, with the exception of the sequences for CCUG 10373 and

LMG 10210, which are 541 and 549 bp in length, respectively. Bootstrap values (%) are indicated at the nodes (500 replicates).

L. Debruyne et al. / Systematic and Applied Microbiology 33 (2010) 7–1412

subgroup 1 strains that were comparable to those observedbetween representatives of subgroups 1 and 2, were also observedin the study by Kiehlbauch et al. [24] and are substantiated by thehsp60 gene sequence analysis (Fig. 2). Like for the genus

Campylobacter[22], the hsp60 gene-based phylogeny was con-gruent with the 16S rRNA gene-based phylogeny, but offered anincreased resolution, in particular for the closely related animal-associated species. However, considering the diversity observed

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L. Debruyne et al. / Systematic and Applied Microbiology 33 (2010) 7–14 13

among A. cryaerophilus strains, great care should be taken whenusing this technique for species-level identification.

In conclusion, A. cryaerophilus clearly has a complex taxonomicstructure and consists of multiple cores of strains, which shareintermediate levels of DDH and exhibit low levels of DDH towardsother Arcobacter species [7,15,24,41]. One of these cores (AFLPsubcluster A) consists of the majority of strains and includes mostbut not all subgroup 2 strains previously studied. The type strainis genetically aberrant from all other strains and thus representsstrains belonging to this species very inadequately. Although thegenetic divergence between AFLP subcluster A strains and the A.

cryaerophilus type strain may justify the proposal of a distinctspecies name for the former, we consider this inappropriatepending the availability of additional strains grouping with thetype strain. Furthermore, a reassessment of the biochemicalcharacteristics published earlier [41] did not allow to differentiatethe type strain from the subgroup 2 strains previously studied.The present results thus demonstrate that the current subgroupnomenclature in A. cryaerophilus should be abandoned and that arepresentative strain of AFLP subcluster A (for instance strain LMG10829 [25]) represents the majority of A. cryaerophilus strainsmore adequately then the type strain.

Appendix A. Supplementary materials

Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.syapm.2009.10.001.

References

[1] Atabay, H.I., Waino, M., Madsen, M. (2006) Detection and diversity of variousArcobacter species in Danish poultry. Int. J. Food Microbiol. 109, 139–145.

[2] Blaiotta, G., Fusco, V., Ercolini, D., Aponte, M., Pepe, O., Villani, F. (2008)Lactobacillus strain diversity based on partial hsp60 gene sequences anddesign of PCR-restriction fragment length polymorphism assays for speciesidentification and differentiation. Appl. Environ. Microbiol. 74, 208–215.

[3] Chinivasagam, H.N., Corney, B.G., Wright, L.L., Diallo, I.S., Blackall, P.J. (2007)Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils insoutheast Queensland. J. Appl. Microbiol. 103, 418–426.

[4] L. Collado, I. Cleenwerck, S. Van Trappen, P. De Vos, M.J. Figueras, Arcobactermytili. sp. nov., an indoxyl acetate hydrolysis-negative bacterium isolatedfrom mussels Int. J. Syst. Evol. Microbiol. 59, 1391–1396.

[5] Costas, M., Pot, B., Vandamme, P., Kersters, K., Owen, R.J., Hill, L.R. (1990)Interlaboratory comparative study of the numerical analysis of one-dimen-sional sodium dodecyl sulphate-polyacrylamide gel electrophoretic proteinpatterns of Campylobacter strains. Electrophoresis 11, 467–474.

[6] L. Debruyne, S.L.W. On, E. De Brandt, P. Vandamme, Novel Campylobacter lari-like bacteria from humans and molluscs: description of Campylobacterpeloridis sp. nov., Campylobacter lari subsp. concheus subsp. nov., andCampylobacter lari subsp. lari subsp. nov.., Int. J. Syst. Evol. Microbiol. 59,1126–1132.

[7] Donachie, S.P., Bowman, J.P., On, S.L.W., Alam, M. (2005) Arcobacter halophilussp. nov., the first obligate halophile in the genus Arcobacter. Int. J. Syst. Evol.Microbiol. 55, 1271–1277.

[8] Duim, B., Vandamme, P.A., Rigter, A., Laevens, S., Dijkstra, J.R., Wagenaar, J.A.(2001) Differentiation of Campylobacter species by AFLP fingerprinting.Microbiology 147, 2729–2737.

[9] Duim, B., Wagenaar, J.A., Dijkstra, J.R., Goris, J., Endtz, H.P., Vandamme, P.A.(2004) Identification of distinct Campylobacter lari genogroups by amplifiedfragment length polymorphism and protein electrophoretic profiles. Appl.Environ. Microbiol. 70, 18–24.

[10] Duim, B., Wassenaar, T.M., Rigter, A., Wagenaar, J. (1999) High-resolutiongenotyping of Campylobacter strains isolated from poultry and humans withamplified fragment length polymorphism fingerprinting. Appl. Environ.Microbiol. 65, 2369–2375.

[11] Goh, S.H., Potter, S., Wood, J.O., Hemmingsen, S.M., Reynolds, R.P., Chow, A.W.(1996) HSP60 gene sequences as universal targets for microbial speciesidentification: studies with coagulase-negative staphylococci. J. Clin. Micro-biol. 34, 818–823.

[12] Hill, J.E., Paccagnella, A., Law, K., Melito, P.L., Woodward, D.L., Price, L., Leung,A.H., Ng, L.K., Hemmingsen, S.M., Goh, S.H. (2006) Identification ofCampylobacter spp. and discrimination from Helicobacter and Arcobacterspp. by direct sequencing of PCR-amplified cpn60 sequences and comparison

to cpnDB, a chaperonin reference sequence database. J. Med. Microbiol. 55,393–399.

[13] Hill, J.E., Penny, S.L., Crowell, K.G., Goh, S.H., Hemmingsen, S.M. (2004) cpnDB:a chaperonin sequence database. Genome Res. 14, 1669–1675.

[14] Houf, K., De Zutter, L., Verbeke, B., Van Hoof, J., Vandamme, P. (2003)Molecular characterization of Arcobacter isolates collected in a poultryslaughterhouse. J. Food Prot. 66, 364–369.

[15] Houf, K., On, S.L., Coenye, T., Mast, J., Van Hoof, J., Vandamme, P. (2005)Arcobacter cibarius sp. nov., isolated from broiler carcasses. Int. J. Syst. Evol.Microbiol. 55, 713–717.

[16] Houf, K., On, S.L.W., Coenye, T., Debruyne, L., De Smet, S., Vandamme, P.(2009) Arcobacter thereius sp. nov., isolated from pigs and ducks. Int. J. Syst.Evol. Microbiol. 59, 2599–2604.

[17] Houf, K., Stephan, R. (2007) Isolation and characterization of the emergingfoodborn pathogen Arcobacter from human stool. J. Microbiol. Methods 68,408–413.

[18] Huys, G., Coopman, R., Janssen, P., Kersters, K. (1996) High-resolutiongenotypic analysis of the genus Aeromonas by AFLP fingerprinting. Int. J.Syst. Bacteriol. 46, 572–580.

[19] Janssen, P., Coopman, R., Huys, G., Swings, J., Bleeker, M., Vos, P., Zabeau, M.,Kersters, K. (1996) Evaluation of the DNA fingerprinting method AFLP as annew tool in bacterial taxonomy. Microbiology 142, 1881–1893.

[20] Janssen, P., Maquelin, K., Coopman, R., Tjernberg, I., Bouvet, P., Kersters, K.,Dijkshoorn, L. (1997) Discrimination of Acinetobacter genomic species byAFLP fingerprinting. Int. J. Syst. Bacteriol. 47, 1179–1187.

[21] Kalpoe, J.S., Templeton, K.E., Horrevorts, A.M., Endtz, H.P., Kuijper, E.J.,Bernards, A.T., Klaassen, C.H.W. (2007) Molecular typing of a suspectedcluster of Nocardia farcinica infections by use of randomly amplifiedpolymorphic DNA, pulsed-field gel electrophoresis, and amplified fragmentlength polymorphism analyses. J. Clin. Microbiol. 45, 4048–4050.

[22] Karenlampi, R.I., Tolvanen, T.P., Hanninen, M.L. (2004) Phylogenetic analysisand PCR-restriction fragment length polymorphism identification of Campy-lobacter species based on partial groEL gene sequences. J. Clin. Microbiol. 42,5731–5738.

[23] Keto-Timonen, R., Tolvanen, R., Lunden, J., Korkeala, H. (2007) An 8-yearsurveillance of the diversity and persistence of Listeria monocytogenes in achilled food processing plant analyzed by amplified fragment lengthpolymorphism. J. Food Prot. 70, 1866–1873.

[24] Kiehlbauch, J.A., Brenner, D.J., Nicholson, M.A., Baker, C.N., Patton, C.M.,Steigerwalt, A.G., Wachsmuth, I.K. (1991) Campylobacter butzleri sp. nov.isolated from humans and animals with diarrheal illness. J Clin. Microbiol. 29,376–385.

[25] Kwok, A.Y., Chow, A.W. (2003) Phylogenetic study of Staphylococcus andMacrococcus species based on partial hsp60 gene sequences. Int. J. Syst. Evol.Microbiol. 53, 87–92.

[26] McClung, C.R., Patriquin, D.G., Davis, R.E. (1983) Campylobacter nitrofigilis sp.nov., a nitrogen-fixing bacterium associated with roots of Spartina alternifloraLoisel. Int. J. Syst. Bacteriol. 33, 605–612.

[27] Melles, D.C., van Leeuwen, W.B., Snijders, S.V., Horst-Kreft, D., Peeters, J.K.,Verbrugh, H.A., van Belkum, A. (2007) Comparison of multilocus sequencetyping (MLST), pulsed-field gel electrophoresis (PFGE), and amplifiedfragment length polymorphism (AFLP) for genetic typing of Staphylococcusaureus. J. Microbiol. Methods 69, 371–375.

[28] Neill, S.D., Campbell, J.N., Obrien, J.J., Weatherup, S.T.C., Ellis, W.A. (1985)Taxonomic position of Campylobacter cryaerophila sp. nov. Int. J. Syst.Bacteriol. 35, 342–356.

[29] On, S.L., Harrington, C.S., Atabay, H.I. (2003) Differentiation ofArcobacter species by numerical analysis of AFLP profiles and description ofa novel Arcobacter from pig abortions and turkey faeces. J. Appl. Microbiol. 95,1096–1105.

[30] On, S.L., Jensen, T.K., Bille-Hansen, V., Jorsal, S.E., Vandamme, P. (2002)Prevalence and diversity of Arcobacter spp. isolated from the internal organsof spontaneous porcine abortions in Denmark. Vet. Microbiol. 85, 159–167.

[31] Pitcher, D.G., Saunders, N.A., Owen, R.J. (1989) Rapid extraction of bacterialgenomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151–156.

[32] Pot, B., Vandamme, P., Kersters, K. (1994) Analysis of electrophoretic whole-organism protein fingerprinting. In: Goodfellow, M., O’Donnell, A.G. (Eds.),Modern Microbial Methods: Chemical Methods in Bacterial Systematics.Wiley, Chichester, United Kingdom, pp. 493–521.

[33] Prouzet-Mauleon, V., Labadi, L., Bouges, N., Menard, A., Megraud, F. (2006)Arcobacter butzleri: underestimated enteropathogen. Emerg. Infect. Dis. 12,307–309.

[34] Rademaker, J.L., Hoste, B., Louws, F.J., Kersters, K., Swings, J., Vauterin, L.,Vauterin, P., de Bruijn, F.J. (2000) Comparison of AFLP and rep-PCR genomicfingerprinting with DNA-DNA homology studies: Xanthomonas as a modelsystem. Int. J. Syst. Evol. Microbiol. 50 (Pt 2), 665–677.

[35] Rehm, T., Baums, C.G., Strommenger, B., Beyerbach, M., Valentin-Weigand, P., Goethe, R. (2007) Amplified fragment length poly-morphism of Streptococcus suis strains correlates with their profile ofvirulence-associated genes and clinical background. J. Med. Microbiol. 56,102–109.

[36] Thompson, F.L., Cleenwerck, I., Swings, J., Matsuyama, J., Iida, T. (2007)Genomic diversity and homologous recombination in Vibrio parahaemolyticusas revealed by amplified fragment length polymorphism (AFLP) and multi-locus sequence analysis (MLSA). Microb. Environ. 22, 373–379.

ARTICLE IN PRESS

L. Debruyne et al. / Systematic and Applied Microbiology 33 (2010) 7–1414

[37] Vandamme, P., Falsen, E., Rossau, R., Hoste, B., Segers, P., Tytgat, R., De Ley, J.(1991) Revision of Campylobacter, Helicobacter, and Wolinella taxonomy:emendation of generic descriptions and proposal of Arcobacter gen. nov. Int. J.Syst. Bacteriol. 41, 88–103.

[38] Vandamme, P., Pot, B., Falsen, E., Kersters, K., De Ley, J. (1990) Intra- andinterspecific relationships of veterinary campylobacters revealed by numer-ical analysis of electrophoretic protein profiles and DNA:DNA hybridizations.Syst. Appl. Microbiol. 13, 295–303.

[39] Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, K., Swings, J. (1996)Polyphasic taxonomy, a consensus approach to bacterial systematics.Microbiol. Rev. 60, 407–438.

[40] Vandamme, P., Pugina, P., Benzi, G., Van Etterijck, R., Vlaes, L., Kersters, K.,Butzler, J.P., Lior, H., Lauwers, S. (1992) Outbreak of recurrent abdominalcramps associated with Arcobacter butzleri in an Italian school. J. Clin.Microbiol. 30, 2335–2337.

[41] Vandamme, P., Vancanneyt, M., Pot, B., Mels, L., Hoste, B., Dewettinck, D.,Vlaes, L., Vandenborre, C., Higgins, R., Hommez, J., Kersters, K., Butzler, J.P.,Goossens, H. (1992) Polyphasic taxonomic study of the emended genusArcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp.

nov., an aerotolerant bacterium isolated from veterinary specimens. Int. J.Syst. Bacteriol. 42, 344–356.

[42] O. Vandenberg, A. Dediste, K. Houf, S. Ibekwem, H. Souayah, S. Cadranel, N.Douat, G. Zissis, J.P. Butzler, P. Vandamme, Arcobacter species in humans.

Emerg. Infect. Dis. 10 (2004) 1863–1867.[43] Viale, A.M., Arakaki, A.K., Soncini, F.C., Ferreyra, R.G. (1994) Evolutionary

relationships among eubacterial groups as inferred from GroEL (chaperonin)sequence comparisons. Int. J. Syst. Bacteriol. 44, 527–5333.

[44] Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters,A., Pot, J., Peleman, J., Kuiper, M., et al. (1995) AFLP: a new technique for DNAfingerprinting. Nucleic Acids Res. 23, 4407–4414.

[45] Wirsen, C.O., Sievert, S.M., Cavanaugh, C.M., Molyneaux, S.J., Ahmad, A.,Taylor, L.T., DeLong, E.F., Taylor, C.D. (2002) Characterization of anautotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamen-tous sulfur. Appl. Environ. Microbiol. 68, 316–325.

[46] I. Wybo, J. Breynaert, S. Lauwers, F. Lindenburg, K. Houf, Isolation ofArcobacter skirrowii from a patient with chronic diarrhea. J. Clin. Microbiol.42 (2004) 1851–1852.