BACKGROUND
the genus arcobacter, included in the family campylobacteraceae, has expanded rapidly since it was first recognised in  <dig>  <cit> , and currently includes  <dig> species.
some of these species are considered enteropathogenic to humans and animals, as well as important zoonotic agents.
arcobacter species negatively impact the food industry, as many meat products are frequently contaminated with these bacteria, and multiple species have been described from shellfish  <cit> .
in addition, the international commission on microbiological specification for foods classified a. butzleri as a serious hazard to human health  <cit> .
however, the true incidence of arcobacter species in environmental and clinical samples is thought to be underestimated because specific detection and identification methods are not normally applied and can be inaccurate  <cit> .
a 16s rrna restriction fragment length polymorphism  method for the identification of arcobacter species has previously been described  <cit> .
the method involved a single digestion with the msei endonuclease and discriminated all arcobacter species that had been described up to  <dig>  i.e.
a. butzleri, a. cryaerophilus, a. cibarius, a. skirrowii, a. nitrofigilis and a. halophilus <cit> .
further molecular methods for the identification of arcobacter species have been reviewed elsewhere  <cit> .
most of the methods described target only the most common species i.e.
a. butzleri <cit> , a. cryaerophilus <cit>  and/or a. skirrowii <cit> .
even the most recently proposed identification method, m-pcr, described by douidah et al.
<cit>  in  <dig>  only targeted five species: a. butzleri, a. cryaerophilus, a. skirrowii, a. cibarius and a. thereius.
furthermore, using this method, the species a. defluvii, a. ellisii, a. venerupis and a. butzleri produced an identical and therefore uninformative amplicon  <cit> .
the limitations of the current methods have arisen because of the limited testing of certain species, as well as the identification of novel species  <cit> .
douidah et al.
<cit>  suggested that the reliance of the currently-available 16s rrna-rflp method on polyacrylamide gel electrophoresis was a major disadvantage for its routine use.
furthermore, the recently described species a. thereius, isolated from aborted pig foetuses  <cit> , and a. trophiarum, which was recovered from porcine faecal matter  <cit> , produce the same rflp pattern as a. butzleri <cit> .
additionally, the new species a. venerupis, from clams, produces a pattern that is very similar to a. marinus <cit> .
the aim of the present study was to update the 16s rrna-rflp identification method to include all the currently characterised species of arcobacter, and to provide protocols for both polyacrylamide and agarose gel electrophoresis so that the method can easily be adapted.
RESULTS
msei digestion can discriminate  <dig> of the  <dig> currently described arcobacter species
following digestion with the endonuclease msei, species-specific differential rflp patterns were obtained for  <dig> of the  <dig> strains , representing  <dig> of the  <dig> species that make up the arcobacter genus , including the new described species a. cloacae .
however, a. venerupis produced a pattern very similar to that of a. marinus, with only a single  <dig> bp band distinguishing the two species .
in addition, the new species a. suis  showed the same banding pattern as a. defluvii, while the characteristic a. butzleri pattern  was also observed following msei digestion of a. thereius and a. trophiarum and  <dig> of the  <dig>  a. cryaerophilus strains.
of these, nine strains  were isolated from animal faeces in valdivia, chile, and two strains were isolated in ireland  from aborted ovine and bovine foetuses, respectively.
the rflp results for these  <dig> strains were discordant with those of m-pcr and their identity was confirmed by sequencing the 16s rrna and rpob genes.
atcc american type culture collection, lmg belgian co-ordinated collection of micro-organisms, cect colección española de cultivos tipo.
¶ sequenced 16s rrna gene.
Ω sequenced rpob gene.
microhetergeneities in a. cryaerophilus strains interfere with rflp identification
the chromatograms of the 16s rrna gene sequences  of seven of the  <dig> unresolved a. cryaerophilus strains  showed mutations  at positions  <dig>  and  <dig> , which were within the target region  of the msei endonuclease .
digestion with mnli and/or bfai resolves the remaining species
a second restriction digest using mnli  was then carried out for those strains with common or similar rflp patterns following msei digestion .
mnli generated a species-specific pattern for a. butzleri, a. thereius, a. marinus and a. venerupis, and a common pattern for a. trophiarum and the atypical strains of a. cryaerophilus .
a further restriction digest step using fspbi , an isoschizomer of bfai, produced species-specific rflp patterns for the separation of a. defluvii from a. suis , and a. trophiarum from the atypical a. cryaerophilus strains .
after carrying out 16s rrna gene restriction digests as illustrated in figure  <dig>  all of the  <dig> strains were correctly identified.
discussion
the proposed 16s rrna-rflp method described here used an initial digestion with msei endonuclease, as in the original method  <cit> , which enabled  <dig> of the  <dig> accepted species, including the recently described species a. cloacae, to be identified.
further digestion was necessary to resolve species that showed the msei digestion pattern of a. butzleri .
computer simulation revealed that two endonucleases, mnli and tasi, produced discriminative patterns between the species a. butzleri and a. thereius .
furthermore, these two enzymes also produced discriminative patterns between a. marinus and a. venerupis , which showed distinctive but very similar patterns following msei digestion .
mnli was selected because it generated more distinctive banding patterns, enabling easier discrimination than tasi .
considering that a. butzleri is a very common species  <cit> , the identification of the majority of strains will normally be obtained with this second  endonuclease reaction .
in fact,  <dig> % of the strains  included in the current study were correctly identified with this second digestion step.
however, a third digestion, using the enzyme bfai, was required to distinguish between a. defluvii and the recently described species a. suis and for distinguishing a. trophiarum from the atypical a. cryaerophilus strains following mnli digestion .
the proposed method enables reliable and fast species identification for a large collection of isolates, requiring, at most, digestion of the pcr-amplified 16s rrna gene  with three restriction endonucleases .
the original 16s rrna-rflp method  <cit>  has been used to identify more than  <dig> arcobacter strains recovered from meat products, shellfish and water in various studies  <cit> .
the existing method has also helped to discover new species on the basis of novel rflp patterns, including a. mytili <cit> , a. molluscorum <cit> , a. ellisii <cit> , a. bivalviorum, a. venerupis <cit>  and a. cloacae <cit> .
furthermore, as well as identifying the more common arcobacter species, this technique has confirmed the presence of other rare species in atypical habitats, such a. nitrofigilis in mussels and a. thereius in pork meat  <cit> .
the updated technique described here is likely to supersede the current method in all of these areas.
the use of the 16s rrna-rflp method in parallel with the more commonly used molecular identification method, m-pcr  <cit> , as well as the fact that strains with incongruent results were sequenced , ensured accurate species identification, and highlighted the limitations of both identification methods  <cit> .
the presence of microheterogeneities in the 16s rrna gene, as in the case of the  <dig> atypical a. cryaerophilus strains, had not previously been observed.
these strains produced the m-pcr amplicon expected for a. cryaerophilus, which targets the 23s rrna gene  <cit> , but showed the a. butzleri 16s rrna-rflp pattern  <cit> .
however, rpob and 16s rrna gene sequencing results confirmed these strains as a. cryaerophilus.
16s rrna-rflp patterns that differ from those described here can be expected for any newly discovered arcobacter species  <cit> .
nevertheless, intra-species nucleotide diversity  at the endonuclease cleavage sites can also generate a novel rflp pattern for a given isolate, or result in a pattern identical to another species  <cit> .
in the latter situation, misidentifications may occur, as described here.
CONCLUSIONS
in conclusion, the 16s rrna-rflp protocols described here for the identification of arcobacter spp.
can be carried out using either agarose or polyacrylamide gel electrophoresis , depending on the requirements of an individual laboratory.
it is important, however, to carry out the 16s rrna gene digestions in the order illustrated in the flow chart .
the method provided in this study is reproducible, reliable, simple, fast, and reasonably inexpensive, and can be carried out efficiently in any laboratory.
the technique is highly applicable for investigations of the prevalence of arcobacters in a variety of food products, water, wastewater or other environmental samples.
it will enable investigators to determine the true incidence of the recently described species a. mytili, a. marinus, a. trophiarum, a. molluscorum, a. defluvii, a. ellisii, a. bivalviorum, a. venerupis, a. cloacae and a. suis clarifying their prevalence and epidemiology.

 the genus arcobacter, included in the family campylobacteraceae, has expanded rapidly since it was first recognised in, and currently includes species. some of these species are considered enteropathogenic to humans and animals, as well as important zoonotic agents. arcobacter species negatively impact the food industry, as many meat products are frequently contaminated with these bacteria, and multiple species have been described from shellfish. in addition, the international commission on microbiological specification for foods classified a. butzleri as a serious hazard to human health. however, the true incidence of arcobacter species in environmental and clinical samples is thought to be underestimated because specific detection and identification methods are not normally applied and can be inaccurate. a 16s rrna restriction fragment length polymorphism method for the identification of arcobacter species has previously been described. the method involved a single digestion with the msei endonuclease and discriminated all arcobacter species that had been described up to i.e. a. butzleri, a. cryaerophilus, a. cibarius, a. skirrowii, a. nitrofigilis and a. halophilus. further molecular methods for the identification of arcobacter species have been reviewed elsewhere. most of the methods described target only the most common species i.e. a. butzleri, a. cryaerophilus and/or a. skirrowii. even the most recently proposed identification method, m-pcr, described by douidah et al. in only targeted five species: a. butzleri, a. cryaerophilus, a. skirrowii, a. cibarius and a. thereius. furthermore, using this method, the species a. defluvii, a. ellisii, a. venerupis and a. butzleri produced an identical and therefore uninformative amplicon. the limitations of the current methods have arisen because of the limited testing of certain species, as well as the identification of novel species. douidah et al. suggested that the reliance of the currently-available 16s rrna-rflp method on polyacrylamide gel electrophoresis was a major disadvantage for its routine use. furthermore, the recently described species a. thereius, isolated from aborted pig foetuses, and a. trophiarum, which was recovered from porcine faecal matter, produce the same rflp pattern as a. butzleri. additionally, the new species a. venerupis, from clams, produces a pattern that is very similar to a. marinus. the aim of the present study was to update the 16s rrna-rflp identification method to include all the currently characterised species of arcobacter, and to provide protocols for both polyacrylamide and agarose gel electrophoresis so that the method can easily be adapted.  msei digestion can discriminate of the currently described arcobacter species following digestion with the endonuclease msei, species-specific differential rflp patterns were obtained for of the strains, representing of the species that make up the arcobacter genus, including the new described species a. cloacae. however, a. venerupis produced a pattern very similar to that of a. marinus, with only a single bp band distinguishing the two species. in addition, the new species a. suis showed the same banding pattern as a. defluvii, while the characteristic a. butzleri pattern was also observed following msei digestion of a. thereius and a. trophiarum and of the a. cryaerophilus strains. of these, nine strains were isolated from animal faeces in valdivia, chile, and two strains were isolated in ireland from aborted ovine and bovine foetuses, respectively. the rflp results for these strains were discordant with those of m-pcr and their identity was confirmed by sequencing the 16s rrna and rpob genes. atcc american type culture collection, lmg belgian co-ordinated collection of micro-organisms, cect colección española de cultivos tipo. ¶ sequenced 16s rrna gene. Ω sequenced rpob gene. microhetergeneities in a. cryaerophilus strains interfere with rflp identification the chromatograms of the 16s rrna gene sequences of seven of the unresolved a. cryaerophilus strains showed mutations at positions and, which were within the target region of the msei endonuclease. digestion with mnli and/or bfai resolves the remaining species a second restriction digest using mnli was then carried out for those strains with common or similar rflp patterns following msei digestion. mnli generated a species-specific pattern for a. butzleri, a. thereius, a. marinus and a. venerupis, and a common pattern for a. trophiarum and the atypical strains of a. cryaerophilus. a further restriction digest step using fspbi, an isoschizomer of bfai, produced species-specific rflp patterns for the separation of a. defluvii from a. suis, and a. trophiarum from the atypical a. cryaerophilus strains. after carrying out 16s rrna gene restriction digests as illustrated in figure  all of the strains were correctly identified. discussion the proposed 16s rrna-rflp method described here used an initial digestion with msei endonuclease, as in the original method, which enabled of the accepted species, including the recently described species a. cloacae, to be identified. further digestion was necessary to resolve species that showed the msei digestion pattern of a. butzleri. computer simulation revealed that two endonucleases, mnli and tasi, produced discriminative patterns between the species a. butzleri and a. thereius. furthermore, these two enzymes also produced discriminative patterns between a. marinus and a. venerupis, which showed distinctive but very similar patterns following msei digestion. mnli was selected because it generated more distinctive banding patterns, enabling easier discrimination than tasi. considering that a. butzleri is a very common species, the identification of the majority of strains will normally be obtained with this second endonuclease reaction. in fact, % of the strains included in the current study were correctly identified with this second digestion step. however, a third digestion, using the enzyme bfai, was required to distinguish between a. defluvii and the recently described species a. suis and for distinguishing a. trophiarum from the atypical a. cryaerophilus strains following mnli digestion. the proposed method enables reliable and fast species identification for a large collection of isolates, requiring, at most, digestion of the pcr-amplified 16s rrna gene with three restriction endonucleases. the original 16s rrna-rflp method has been used to identify more than arcobacter strains recovered from meat products, shellfish and water in various studies. the existing method has also helped to discover new species on the basis of novel rflp patterns, including a. mytili, a. molluscorum, a. ellisii, a. bivalviorum, a. venerupis and a. cloacae. furthermore, as well as identifying the more common arcobacter species, this technique has confirmed the presence of other rare species in atypical habitats, such a. nitrofigilis in mussels and a. thereius in pork meat. the updated technique described here is likely to supersede the current method in all of these areas. the use of the 16s rrna-rflp method in parallel with the more commonly used molecular identification method, m-pcr, as well as the fact that strains with incongruent results were sequenced, ensured accurate species identification, and highlighted the limitations of both identification methods. the presence of microheterogeneities in the 16s rrna gene, as in the case of the atypical a. cryaerophilus strains, had not previously been observed. these strains produced the m-pcr amplicon expected for a. cryaerophilus, which targets the 23s rrna gene, but showed the a. butzleri 16s rrna-rflp pattern. however, rpob and 16s rrna gene sequencing results confirmed these strains as a. cryaerophilus. 16s rrna-rflp patterns that differ from those described here can be expected for any newly discovered arcobacter species. nevertheless, intra-species nucleotide diversity at the endonuclease cleavage sites can also generate a novel rflp pattern for a given isolate, or result in a pattern identical to another species. in the latter situation, misidentifications may occur, as described here.  in conclusion, the 16s rrna-rflp protocols described here for the identification of arcobacter spp. can be carried out using either agarose or polyacrylamide gel electrophoresis, depending on the requirements of an individual laboratory. it is important, however, to carry out the 16s rrna gene digestions in the order illustrated in the flow chart. the method provided in this study is reproducible, reliable, simple, fast, and reasonably inexpensive, and can be carried out efficiently in any laboratory. the technique is highly applicable for investigations of the prevalence of arcobacters in a variety of food products, water, wastewater or other environmental samples. it will enable investigators to determine the true incidence of the recently described species a. mytili, a. marinus, a. trophiarum, a. molluscorum, a. defluvii, a. ellisii, a. bivalviorum, a. venerupis, a. cloacae and a. suis clarifying their prevalence and epidemiology. 
digestion of the 16s rrna gene with the endonuclease msei produced clear, distinctive patterns for of the species, while the remaining species shared a common or very similar rflp pattern. the number of characterised arcobacter species has since risen to the aim of the present study was to update the 16s rrna-rflp identification method to include all currently characterised species of arcobacter. using either polyacrylamide or agarose gel electrophoresis. microheterogeneities within the 16s rrna gene, which interfered with the rflp identification, were also documented for the first time in this genus, particularly in strains of arcobacter cryaerophilus isolated from animal faeces and aborted foetuses. subsequent digestion of the 16s rrna gene from these species with the endonucleases mnli and/or bfai generated species-specific rflp patterns. a previously published 16s rrna-rflp arcobacter spp. 16s rrna-rflp analysis identified arcobacter spp. identification method produced specific rflp patterns for the six species described at that time, using a single endonuclease.
 arcobacter spp. are ubiquitous zoonotic bacteria that are being increasingly recognised as a threat to human health. a previously published 16s rrna-rflp arcobacter spp. identification method produced specific rflp patterns for the six species described at that time, using a single endonuclease. the number of characterised arcobacter species has since risen to the aim of the present study was to update the 16s rrna-rflp identification method to include all currently characterised species of arcobacter.  digestion of the 16s rrna gene with the endonuclease msei produced clear, distinctive patterns for of the species, while the remaining species shared a common or very similar rflp pattern. subsequent digestion of the 16s rrna gene from these species with the endonucleases mnli and/or bfai generated species-specific rflp patterns.  16s rrna-rflp analysis identified arcobacter spp. using either polyacrylamide or agarose gel electrophoresis. microheterogeneities within the 16s rrna gene, which interfered with the rflp identification, were also documented for the first time in this genus, particularly in strains of arcobacter cryaerophilus isolated from animal faeces and aborted foetuses. arcobacteridentificationagarosepolyacrylamide16s rrna-rflp16s rrna gene mutations 