Towards a typing strategy for Arcobacter species isolated from humans and animals and assessment of the in vitro genomic stability

Arcobacter species have a widespread distribution with a broad range of animal hosts and environmental reservoirs, and are increasingly associated with human illness. To elucidate the routes of infection, several characterization methods such as pulsed-field gel electrophoresis (PFGE), amplified fragment-length polymorphism, and enterobacterial repetitive intergenic consensus (ERIC)-PCR have already been applied, but without proper validation or comparison. At present, no criterion standard typing method or strategy has been proposed. Therefore, after the validation of PFGE, those commonly applied typing methods were compared for the characterization of six human- and animal-associated Arcobacter species. With a limited number of isolates to be characterized, PFGE with restriction by KpnI is proposed as the first method of choice. However, ERIC-PCR represents a more convenient genomic fingerprinting technique when a large number of isolates is involved. Therefore, a first clustering of similar patterns obtained after ERIC-PCR, with a subsequent typing of some representatives per ERIC cluster by PFGE, is recommended. As multiple genotypes are commonly isolated from the same host and food, genomic plasticity has been suggested. The in vitro genomic stability of Arcobacter butzleri and A. cryaerophilus was assessed under two temperatures and two oxygen concentrations. Variability in the genomic profile of A. cryaerophilus was observed after different passages for different strains at 37 degrees C under microaerobic conditions. The bias due to these genomic changes must be taken into account in the evaluation of the relationship of strains

Presence and analysis of plasmids in human and animal associated Arcobacter species

In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration

Alternative sampling to establish the Escherichia coli O157 status on beef cattle farms

Prevalence of Escherichia coli O157 in cattle at the farm level is mostly determined by taking individually rectal samples. From the animal welfare point of view the collection of such samples on the farm is not advisable. The present study evaluated alternative sample types to assess the E. coli O157 status of cattle farms. Twelve closed cattle farms were visited twice with a time interval of 6-8 months. Rectal and hide surface samples (the nose, the neck, the shoulder, the flank, and the round) were collected from beef cattle within the period of 5 months before slaughter and from their environment (overshoes from the pen bedding, swabs from the pen barrier, feed and water). Statistical analysis revealed that from all samples taken only the "overshoe method" might be a good sampling technique to substitute the collection of individual fecal samples to establish the E. coli O157 status of a farm and even a pen. Characterization of the isolates, using pulsed field gel electrophoresis, revealed that on each positive farm only one genotype was presented, even after a period of more than 6 months

Composition and digestibility of whole plant triticale as affected by maturity stage and genotype

Maize is grown in monoculture on many dairy farms in NW Europe. Triticale for use as whole plant (WPT) may contribute to the diversification of crop rotation, resulting in a lower disease and weed pressure and higher yields. Moreover with climate changing to warmer and dryer summers, WPT offers an advantage compared with maize as it can be harvested before the summer heat. A six-year study was started to investigate the potential of WPT as alternative roughage crop for maize silage. An important objective is to study the factors affecting the digestibility of organic matter (OMd) and cell wall digestibility (NDFd). In a first field trial, 36 genotypes were harvested at 5 maturity stages and samples from these 180 WPT objects were scanned by NIRS. Based on spectral variation, 80 samples were selected to determine dry matter (DM), crude protein (CP), crude fat, crude ash, sugars, starch, NDF, ADF and ADL as well as cellulase OMd and rumen fluid NDFd. The DM content of the WPT for the 5 stages averaged 268, 285, 467, 508 and 574 g/kg, respectively. During maturing, sugar content decreased, whereas starch content increased with a clear shift from the second to the third stage. With advancing maturity there was a relative small decrease in NDF, CP and ash content. The NDFd seemed hardly affected by maturity stage with on average 53.9 and 55.4% for the first and fifth stage. On the other hand, mean OMd increased linearly with later harvesting and amounted to 63.8, 65.0, 67.9, 68.1 and 69.1%, respectively. In addition to the effect of maturity stage, there appeared a large variation among genotypes. For the last stage, OMd ranged between 67.3 and 73.2%. This variation opens perspectives to select for WPT with high nutritive value. The reference data from this first trial were used to develop NIRS calibrations in order to allow rapid feed evaluation throughout the study

Arcobacter trophiarum sp. nov., isolated from fattening pigs

In the course of a longitudinal study elucidating the dynamics of Arcobacter populations in pigs, 16 isolates of Gram-reaction-negative, rod-shaped, slightly curved, non-spore-forming bacteria were grouped by amplified fragment length polymorphism analysis into a distinct phenon within the genus Arcobacter. Fragments were generated for all isolates in a genus-specific PCR assay, but no amplicon was obtained in a species-specific multiplex-PCR test. Numerical analysis of the whole-cell protein profiles also showed that all isolates clustered in a single group that was distinct from related members of the genus Arcobacter. DNA-DNA hybridizations between two representative strains, designated 64(T) and 122, of the isolates obtained exhibited a mean DNA-DNA relatedness of 72%. DNA-DNA hybridizations between strains 64(T) and 122 and reference strains of other animal-related bacteria of the genus Arcobacter revealed binding values of 47% or less. The DNA G + C contents of the two representative strains were 28.5 and 28.4 mol%, respectively, and analysis of three marker genes identified Arcobacter cryaerophilus, A. thereius, A. cibarius and A. skirrowii as their closest phylogenetic neighbours. Strains 64(T) and 122 could be distinguished from other members of the genus Arcobacter by means of biochemical tests for catalase and urease activities, nitrate reduction, indoxyl acetate hydrolysis, lack of growth at 37 degrees C, growth in 2% (w/v) NaCl, growth on 0.1% sodium deoxycholate and non-supplemented Campylobacter charcoal-deoxycholate base medium and resistance to cephalothin (32 mg l(-1)) and cefoperazone (64 mg l(-1)). Additionally, a PCR assay was developed for the detection and identification of strains 64(T) and 122, which represent a novel species of the genus Arcobacter, for which the name Arcobacter trophiarum sp. nov. is proposed. The type strain is strain 64(T) (=LMG 25534(T) =CCUG 59229(T))