Identification of campylobacteria isolated from Danish broilers by phenotypic tests and species-specific PCR assays.

AIMS: To validate a phenotypic Campylobacter species identification method employed to identify campylobacters in broilers by comparison with campylobacterial species identification using various species-specific PCR analyses. METHODS AND RESULTS: From a collection of 2733 phenotypically identified campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while other species identified were Helicobacter pullorum, Camp. lari and Camp. upsaliensis. However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged to H. pullorum. It was also notable that 2-6% of the cultures were, in fact, mixed cultures. CONCLUSIONS: The phenotypic identification scheme employed failed to appropriately differentiate Campylobacter species and particularly to identify the closely related species, H. pullorum. SIGNIFICANCE AND IMPACT OF THE STUDY: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers.

Evaluation of PCR for detection of Campylobacter in a national broiler surveillance programme in Denmark.

AIMS: To develop and evaluate a rapid and sensitive PCR method for detection of Campylobacter spp. directly from chicken faeces. METHODS AND RESULTS: DNA was isolated from faecal swabs using magnetic beads followed by PCR using a prealiquoted PCR mixture, which had been stored in the freezer. The result could be obtained in <6 h. The method was evaluated on 1282 samples from the Danish surveillance programme for Campylobacter in broilers by comparing with conventional culture. The diagnostic specificity was calculated to be 0.99. The detection limits of the PCR method and of the conventional culture were compared using spiked control material. For both methods the detection limit was 36 CFU ml-1. CONCLUSIONS: It was concluded that the PCR proved useful for detection of Campylobacter in pooled cloacal swabs from broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: By taking cloacal samples in the broiler flocks the technique can be used as an important tool for planning and directing the broiler slaughtering process. This will be a great help in minimizing the risk of contaminating Campylobacter-free flocks at the abattoir.

Halorhabdus utahensis gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea from Great Salt Lake, Utah.

Strain AX-2T (T = type strain) was isolated from sediment of Great Salt Lake, Utah, USA. Optimal salinity for growth was 27% (w/v) NaCl and only a few carbohydrates supported growth of the strain. Strain AX-2T did not grow on complex substrates such as yeast extract or peptone. 16S rRNA analysis revealed that strain AX-2T was a member of the phyletic group defined by the family Halobacteriaceae, but there was a low degree of similarity to other members of this family. The polar lipid composition comprising phosphatidyl glycerol, the methylated derivative of diphosphatidyl glycerol, triglycosyl diethers and sulfated triglycosyl diethers, but not phosphatidyl glycerosulfate, was not identical to that of any other aerobic, halophilic species. On the basis of the data presented, it is proposed that strain AX-2T should be placed in a new taxon, for which the name Halorhabdus utahensis is appropriate. The type strain is strain AX-2T (= DSM 12940T).

Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov.

A Gram-positive, extremely halotolerant bacterium was isolated from the Great Salt Lake, Utah, USA. The strain, designated NNT (= DSM 11805T), was strictly aerobic, rod-shaped, motile by peritrichous flagella and spore-forming. Strain NNT grew at salinities of 0-20% (w/v) NaCl. A distinctive feature of strain NNT was its optimal growth in salt-free medium. The polar lipid pattern of strain NNT consisted of phosphatidyl glycerol, diphosphatidyl glycerol and two phospholipids of unknown structure. The G + C content of its DNA was 38 mol%. The morphological, physiological and, particularly, the 16S rDNA sequence data, showed that strain NNT was associated with 'Bacillus group 1'. However, the organisms showing the greatest degree of sequence similarity to strain NNT were members of the genus Halobacillus and the species Marinococcus albus, Virgibacillus pantothenticus, Bacillus salexigens and Bacillus dipsosauri. On the basis of chemotaxonomic data, strain NNT was shown to be chemically most similar to B. salexigens and B. dipsosauri, with the greatest degree of similarity being shown to the latter organism. This was consistent with the 16S rDNA sequence data. Members of the genus Halobacillus comprise a chemically distinct group and can easily be distinguished from all other organisms of 'Bacillus group 1'. On the basis of the 16S rDNA data, chemotaxonomy and the physiology of strain NNT, it is proposed that this organism is a member of a new species, within a new genus, for which the name Gracilibacillus halotolerans is proposed. It is also proposed that B. dipsosauri be transferred to this genus as Gracilibacillus dipsosauri comb. nov. and that B. salexigens be transferred to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. Finally, additional data is provided to support the transfer of Bacillus pantothenticus to the genus Virgibacillus, as Virgibacillus pantothenticus Heyndrickx et al. (1998).