Luis Collado

Taxonomy, Epidemiology, and Clinical Relevance of the Genus Arcobacter

SUMMARY The genus Arcobacter, defined almost 20 years ago from members of the genus Campylobacter, has become increasingly important because its members are being considered emergent enteropathogens and/or potential zoonotic agents. Over recent years information that is relevant for microbiologists, especially those working in the medical and veterinary fields and in the food safety sector, has accumulated. Recently, the genus has been enlarged with several new species. The complete genomes of Arcobacter butzleri and Arcobacter nitrofigilis are available, with the former revealing diverse pathways characteristic of free-living microbes and virulence genes homologous to those of Campylobacter. The first multilocus sequence typing analysis showed a great diversity of sequence types, with no association with specific hosts or geographical regions. Advances in detection and identification techniques, mostly based on molecular methods, have been made. These microbes have been associated with water outbreaks and with indicators of fecal pollution, with food products and water as the suspected routes of transmission. This review updates this knowledge and provides the most recent data on the taxonomy, species diversity, methods of detection, and identification of these microbes as well as on their virulence potential and implication in human and animal diseases.

Presence of Arcobacter spp. in environmental waters correlates with high levels of fecal pollution

We investigated the presence of Arcobacter spp. in 205 water samples of freshwater, seawater and sewage in Spain. These bacteria were present in 55.1% of the samples (113/205) and were significantly associated for the first time with bacterial indicators of fecal pollution. The dominant species in the positive samples was Arcobacter butzleri (94%) followed by Arcobacter cryaerophilus (30%) and Arcobacter skirrowii (1.8%).

Arcobacter mytili sp. nov., an indoxyl acetate-hydrolysis-negative bacterium isolated from mussels

Three Arcobacter isolates, recovered from mussels (genus Mytilus), and one isolate from brackish water in Catalonia (north-east Spain) showed a novel pattern using a recently described identification method for members of the genus Arcobacter, 16S rRNA gene RFLP. Enterobacterial repetitive intergenic consensus PCR fingerprinting demonstrated that the three isolates from mussels belonged to two genotypes and that the fourth isolate from water belonged to a third genotype. Analysis of the 16S rRNA and rpoB gene sequences showed that the new isolates formed a separate lineage within the genus Arcobacter. This was also confirmed by the low DNA-DNA relatedness values (16-30 %) of the isolates with the type strains of recognized Arcobacter species. Hydrolysis of indoxyl acetate, a characteristic trait for all species of the genus Arcobacter, was negative for the novel isolates. The susceptibility of the novel isolates to cefoperazone, together with the lack of urease production and nitrate reduction, further enabled them to be differentiated from recognized Arcobacter species based on physiological characteristics. Genotypic and phenotypic characteristics indicated that the new isolates represent a novel species of the genus Arcobacter, for which the name Arcobacter mytili sp. nov. is proposed, with the type strain F2075(T) (=CECT 7386(T) =LMG 24559(T)). The DNA G+C content of strain F2075(T) was 26.9 mol%.

Prevalence of Arcobacter in Meat and Shellfish

Arcobacter is considered an emergent foodborne and waterborne enteropathogen. However, its prevalence in foods of animal origin is only partially known, because most studies have been concentrated on poultry, pork, and beef, and methods applied do not allow identification of all currently accepted Arcobacter species. We investigated the prevalence of Arcobacter in 203 food samples, 119 samples of seven different types of meats and 84 samples of four types of shellfish. Isolates were identified in parallel by using a published multiplex PCR method and a recently described 16S rDNA restriction fragment length polymorphism method that allows all currently accepted Arcobacter species to be characterized. The global prevalence of Arcobacter was 32%; it was highest in clams (5 of 5 samples, 100%) and chicken (9 of 14 samples, 64.3%) followed by pork (9 of 17 samples, 53.0%), mussels (23 of 56 samples, 41.1%), and duck meat (2 of 5 samples, 40.0%). Turkey meat and beef had a similar recovery rate (10 of 30 samples, 33.3%; 5 of 16 samples, 31.3%; respectively), and rabbit meat had the lowest rate (1 of 10 samples, 10.0%). No arcobacters were found in oysters, frozen shrimps, or sausages. This food survey is the first in which five of the seven accepted Arcobacter species have been isolated. Arcobacter butzleri was the most prevalent species (63.0% of isolates) followed by Arcobacter cryaerophilus (26.6%), Arcobacter mytili (4.7%), Arcobacter skirrowii (3.1%), and Arcobacter nitrofigilis (3.1%). Three (4.7%) of the isolates were classified as belonging to three potentially new phylogenetic lines. Our results indicated that Arcobacter species are widely distributed in the food products studied.

Arcobacter ellisii sp. nov., isolated from mussels

As part of a study carried out for detecting Arcobacter spp. in shellfish, three mussel isolates that were Gram-negative slightly curved rods, non-spore forming, showed a new 16S rDNA-RFLP pattern with a specific identification method for the species of this genus. Sequences of the 16S rRNA gene and those of the housekeeping genes rpoB, gyrB and hsp60 provided evidence that these mussel strains belonged to an unknown genetic lineage within the genus Arcobacter. The similarity between the 16S rRNA gene sequence of the representative strain (F79-6(T)) and type strains of the other Arcobacter species ranged between 94.1% with A. halophilus and 99.1% with the recently proposed species A. defluvii (CECT 7697(T)). DDH results between strain F79-6(T) and the type strain of the latter species were below 70% (53±3.0%). Phenotypic characteristics together with MALDITOF mass spectra differentiated the new mussel strains from all other Arcobacter species. All the results indicate that these strains represent a new species, for which the name Arcobacter ellisii sp. nov. with the type strain F79-6(T) (=CECT 7837(T)=LMG 26155(T)) is proposed.

Arcobacter cloacae sp. nov. and Arcobacter suis sp. nov., two new species isolated from food and sewage

Three strains recovered from mussels (F26), sewage (SW28-13(T)) and pork meat (F41(T)) were characterized as Arcobacter. They did not appear to resemble any known species on the basis of their 16S rDNA-RFLP patterns and the rpoB gene analyses. However, strains F26 and SW28-13(T) appeared to be the same species. The 16S rRNA gene sequence similarity of strains SW28-13(T) and F41(T) to the type strains of all other Arcobacter species ranged from 94.1% to 99.6% and 93.4% to 98.8%, respectively. Phenotypic characteristics and the DNA-DNA hybridization (DDH) results showed that they belonged to 2 new Arcobacter species. A multilocus phylogenetic analysis (MLPA) with the concatenated sequences of 5 housekeeping genes (gyrA, atpA, rpoB, gyrB and hsp60) was used for the first time in the genus, showing concordance with the 16S rRNA gene phylogenetic analysis and DDH results. The MALDI-TOF mass spectra also discriminated these strains as two new species. The names proposed for them are Arcobacter cloacae with the type strain SW28-13(T) (=CECT 7834(T)=LMG 26153(T)) and Arcobacter suis with the type strain F41(T) (=CECT 7833(T)=LMG 26152(T)).

Arcobacter defluvii sp. nov., isolated from sewage samples

A study employing a polyphasic taxonomic approach was undertaken to clarify the position of 12 isolates recovered from sewage samples. These isolates were recognized as a potential novel species because a new and specific pattern was produced with the 16S rRNA-RFLP Arcobacter identification method. The sequences of the 16S rRNA gene not only supported the classification of these novel strains as members of the genus Arcobacter, but also showed that they formed a separate phylogenetic line. Strain SW28-11(T), chosen as the representative of these strains, showed 16S rRNA gene sequence similarity of 95.6 % with the closest related species Arcobacter nitrofigilis. The phylogenetic position of the novel strains was further confirmed by analysis of the housekeeping genes hsp60, rpoB and, for the first time, gyrB. The latter proved to be an excellent additional gene for establishing the phylogeny of this genus. These data, together with phenotypic characterization, revealed that this group of isolates represent a novel species of the genus Arcobacter. The name Arcobacter defluvii sp. nov., is proposed, with the type strain SW28-11(T) ( = CECT 7697(T) = LMG 25694(T)).

Arcobacter molluscorum sp. nov., a new species isolated from shellfish.

Nineteen bacteria isolates recovered from shellfish samples (mussels and oysters) showed a new and specific 16S rDNA-RFLP pattern with an Arcobacter identification method designed to recognize all species described up to 2008. These results suggested that they could belong to a new species. ERIC-PCR revealed that the 19 isolates belonged to 3 different strains. The sequence of the 16S rRNA gene of a representative strain (F98-3(T)) showed 97.6% similarity with the closest species Arcobacter marinus followed by Arcobacter halophilus (95.6%) and Arcobacter mytili (94.7%). The phylogenetic analysis with the16S rRNA, rpoB, gyrB and hsp60 genes placed the shellfish strains within the same cluster as the three species mentioned (also isolated from saline habitats) but they formed an independent phylogenetic line. The DDH results between strain F98-3(T) and A. marinus (54.8%±1.05), confirmed that it represents a new species. Several biochemical tests differentiated the shellfish isolates from all other Arcobacter species. Although the new species was different from A. mytili, they shared not only the same habitat (mussels) but also the characteristic of being so far the only Arcobacter species that are simultaneously negative for urea and indoxyl acetate hydrolysis. All results supported the classification of the shellfish strains as a new species, for which the name Arcobacter molluscorum sp. nov. with the type strain F98-3(T) is proposed (=CECT 7696(T)=LMG 25693(T)).

Occurrence and diversity of Arcobacter spp. along the Llobregat River catchment, at sewage effluents and in a drinking water treatment plant

The presence of Arcobacter species in faecally contaminated environmental waters has previously been studied. However, the ability to eliminate Arcobacter during the water treatment processes that produce drinking water has been little studied. We have investigated the prevalence and diversity of Arcobacter spp. throughout the year at 12 sampling points in the Llobregat River catchment (Catalonia, Spain) including 3 sites at a drinking water treatment plant. Positive samples for Arcobacter spp., came predominantly from the most faecally polluted sites. Recovery rates from all sites were greater in the spring (91.7%) and summer (83.3%) than in autumn and winter (75.0% in both cases), but this trend was not statistically evaluated due to the limited number of samples. Among the 339 colonies analyzed, the most prevalent species by multiplex PCR and 16S rDNA restriction fragment length polymorphism were Arcobacter butzleri (80.2%), followed by Arcobacter cryaerophilus (19.4%) and Arcobacter skirrowii (0.3%). Isolates showed a high genotype diversity as determined by the enterobacterial repetitive intergenic consensus PCR. In fact, 91.2% (309/339) of the colonies had different genotypes, i.e. 248 of them among the 275 isolates of A. butzleri and 60 among the 63 isolates of A. cryaerophilus and 1 genotype of A. skirrowii. Arcobacter was never detected or isolated from finished drinking water, demonstrating that water treatment is effective in removing Arcobacter species.

Updated 16S rRNA-RFLP method for the identification of all currently characterised Arcobacter spp

BackgroundArcobacter spp. (family Campylobacteraceae) 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 (MseI). The number of characterised Arcobacter species has since risen to 17. The aim of the present study was to update the 16S rRNA-RFLP identification method to include all currently characterised species of Arcobacter.ResultsDigestion of the 16S rRNA gene with the endonuclease MseI produced clear, distinctive patterns for 10 of the 17 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.Conclusions16S rRNA-RFLP analysis identified 17 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.