No abstract

A previously developed Arcobacter isolation protocol for poultry skin and meat was validated for the isolation of Arcobacter from feces of livestock animals. Good repeatability, in-lab reproducibility and sensitivity were achieved and the specificity was improved by additional incorporation of cycloheximide and increase of the novobiocin concentration in the selective supplement. The limit of detection of quantitative and qualitative analysis was 10(2) and 10(0) cfu g(-1) feces, respectively. From fecal samples collected at slaughterhouse, Arcobacter was isolated from 43.9% of porcine, 39.2% of bovine, 16.1% of ovine and 15.4% of equine samples. All three animal-associated Arcobacter species were isolated and levels up to 10(3) cfu g(-1) feces were determined.

In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n ‫؍‬ 182) and A. cryaerophilus (n ‫؍‬ 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.

A multiplex PCR assay with five primers targeting the 16S and 23S rRNA genes was developed for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. The selected primers amplify a 257-bp fragment from A. cryaerophilus, a 401-bp fragment from A. butzleri and a 641-bp fragment from A. skirrowii. No PCR product was generated for closely related bacteria including Campylobacter and Helicobacter species. The assay was useful to identify cultures after in vitro cultivation and to detect and identify A. butlzeri and A. cryaerophilus from poultry samples present in 24-h old enrichment in Arcobacter broth with cefoperazone, amphotericin and teicoplanin (CAT)-supplement.

Twenty Gram-negative, rod-shaped, slightly curved, non-spore-forming bacteria that gave a negative result in Arcobacter species-specific PCR tests but that yielded an amplicon in an Arcobacter genus-specific PCR test were isolated from 13 unrelated broiler carcasses. Numerical analysis of the profiles obtained by SDS-PAGE of whole-cell proteins clustered all isolates in a single group distinct from the other Arcobacter species. DNA-DNA hybridization among four representative strains exhibited DNA binding values above 91 %. DNA-DNA hybridization with reference strains of the current four Arcobacter species revealed binding levels below 47 %. The G+C contents ranged between 26?8 and 27?3 mol%. Pairwise comparison of 16S rRNA gene sequences revealed the mean values for similarity to the type strain of Arcobacter cryaerophilus (97?5 %), Arcobacter butzleri (96?5 %), Arcobacter skirrowii (96?0 %) and Arcobacter nitrofigilis (95?0 %). The levels of similarity to Campylobacter and Helicobacter species were below 88 and 87 %, respectively. The isolates could be distinguished from other Arcobacter species by the following biochemical tests: catalase, oxidase and urease activities; reduction of nitrate; growth at 25 and 37 6C under aerobic conditions; growth on 2-4 % (w/v) NaCl media; and susceptibility to cephalothin. These data demonstrate that the 20 isolates represent a single novel Arcobacter species, for which the name Arcobacter cibarius sp. nov. is proposed, with LMG 21996 T (=CCUG 48482 T ) as the type strain.