Advancement of DNA sequencing technology allows the routine use of genome sequences in the various fields of microbiology. The information held in genome sequences proved to provide objective and reliable means in the taxonomy of prokaryotes. Here, we describe the minimal standards for the quality of genome sequences and how they can be applied for taxonomic purposes.

Sequences of the gene encoding theβ-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the familyPasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universalrpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of therpoBgene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with therpoBsequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases therpoBphylogeny was in better agreement with DNA–DNA hybridization studies than the phylogeny derived from 16S rDNA. TherpoBgene is strongly conserved within the various species of the family ofPasteurellaceae. Hence,rpoBgene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of thePasteurellaceaeand may also prove useful for reorganizing the current taxonomy of this bacterial family.

This paper describes a phenotypic and genotypic investigation of the taxonomy of [Haemophilus]paragallinarum,Pasteurella gallinarum,Pasteurella aviumandPasteurella volantium, a major subcluster within the avian 16S rRNA cluster 18 of the familyPasteurellaceae. An extended phenotypic characterization was performed of the type strain of [Haemophilus]paragallinarum, which is NAD-dependent, and eight NAD-independent strains of [Haemophilus]paragallinarum. Complete 16S rRNA gene sequences were obtained for one NAD-independent and four NAD-dependent [Haemophilus]paragallinarumstrains. These five sequences along with existing 16S rRNA gene sequences for 11 other taxa within avian 16S rRNA cluster 18 as well as seven other taxa from thePasteurellaceaewere subjected to phylogenetic analysis. The analysis demonstrated that [Haemophilus]paragallinarum,Pasteurella gallinarum,Pasteurella aviumandPasteurella volantiumformed a monophyletic group with a minimum of 96·8 % sequence similarity. This group can also be separated by phenotypic testing from all other recognized and named taxa within thePasteurellaceae. As both genotypic and phenotypic testing support the separate and distinct nature of this subcluster, the transfer is proposed ofPasteurella gallinarum, [Haemophilus]paragallinarum,Pasteurella aviumandPasteurella volantiumto a new genusAvibacteriumasAvibacterium gallinarumgen. nov., comb. nov.,Avibacterium paragallinarumcomb. nov.,Avibacterium aviumcomb. nov. andAvibacterium volantiumcomb. nov. The type strains are NCTC 1118^T(Avibacterium gallinarum), NCTC 11296^T(Avibacterium paragallinarum), NCTC 11297^T(Avibacterium avium) and NCTC 3438^T(Avibacterium volantium). Key characteristics that separate these four species are catalase activity (absent only inAvibacterium paragallinarum) and production of acid from galactose (negative only inAvibacterium paragallinarum), maltose (negative only inAvibacterium avium) and mannitol (negative inAvibacterium gallinarumandAvibacterium avium).

Objectives: To elucidate the diversity and evolutionary history of plasmid- and chromosomally-located blaZ, to detect indications of frequent exchange of blaZ between human and bovine staphylococci and to estimate the frequency of transfer of blaZ between coagulase-negative staphylococci (CoNS) and Staphylococcus aureus of bovine origin. Methods: blaZ was detected in 143 strains of penicillin-resistant S. aureus and CoNS from five Danish cattle herds (n = 25/23), random CoNS isolates from Denmark (n = 37), a collection of S. aureus from six different countries (n = 52), humans in Denmark (n = 3) and β-lactamase control strains (n = 3). The sequence was determined in 105 strains and compared to published sequences by pairwise and multiple alignments. Maximum likelihood analysis was performed including bootstrap analysis. Parsimony, neighbour joining and consensus comparisons were performed for recombination. The localization of blaZ was determined by Southern blotting in 108 isolates. Results: All penicillin-resistant strains carried blaZ and showed a similar organization of blaR1 and blaZ. The blaZ gene was localized to a plasmid in only 16 of the resistant strains. Sixty-nine sequences representing 105 isolates and sequences retrieved from public databases were compared. A phylogenetic tree showed that blaZ exists in three evolutionary lines: one group was of plasmid origin, one group was of chromosomal origin and one intermediate group. Sixty-nine sequence types were demonstrated. They translated into 11 BlaZ protein types. The major types all contained strains of both human and bovine origin, and more than one Staphylococcus species, demonstrating a shared gene pool. In a comparison of S. aureus and CoNS obtained from five Danish cattle herds, the same type of blaZ was only detected in one case. Conclusions: Results indicated a separate evolution for plasmid- and chromosomally-encoded blaZ. Although a common gene pool seems to exist among staphylococci, exchange of blaZ between strains and species is judged to be an extremely rare event.

The present study was aimed at reducing the time and labour used to perform DNA-DNA hybridizations for classification of bacteria at the species level. A micro-well-format DNA hybridization method was developed and validated. DNA extractions were performed by a small-scale method and DNA was sheared mechanically into fragments of between 400 and 700 bases. The hybridization conditions were calibrated according to DNA similarities obtained by the spectrophotometric method using strains within the family Pasteurellaceae. Optimal conditions were obtained with 300 ng DNA added per well and bound by covalent attachment to NucleoLink. Hybridization was performed with 500 ng DNA, 5% (w/w) of which was labelled with photo-activatable biotin (competitive hybridization) for 2.5 h at 65 degrees C in 2 x SSC followed by stringent washing with 2 x SSC at the same temperature. The criteria for acceptance of results were a maximum of 15% standard deviation, calculated as a percentage of the mean for four replicate micro-wells, and that DNA similarities were not significantly different in at least two independent experiments. The relationship between DNA similarities obtained by the micro-well method (y) and by the spectrophotometric method (x) was y = 0.534x+30.6, when these criteria had been applied to 23 pairs of strains of Actinobacillus species, avian [Pasteurella] haemolytica-like bacteria and Mannheimia species. The correlation (Pearson) between DNA similarities obtained by interchange of strains used for covalent binding and hybridization was 0.794. Significantly lower DNA similarities were observed by the spectrophotometric compared with the micro-well method for three pairs of hybridizations. After removal of these data, the relationship between DNA similarities obtained by the micro-well and spectrophotometric methods improved to y = 0.855x + 11.0. It was found that the accuracy and precision of the micro-well method was at the same level as that of the spectrophotometric method, but the labour and analysis time were reduced significantly. The use of hybridization in the micro-well format will allow DNA-DNA hybridizations to be carried out between all strains selected for a particular taxonomic study, in order to construct complete data matrices and improve species definition.

The microbiota in the human gastrointestinal tract (GIT) is highly exposed to antibiotics, and may be an important reservoir of resistant strains and transferable resistance genes. Maternal GIT strains can be transmitted to the offspring, and resistances could be acquired from birth. This is a case study using a metagenomic approach to determine the diversity of microorganisms conferring tetracycline resistance (Tc^r) in the guts of a healthy mother-infant pair one month after childbirth, and to investigate the potential for horizontal transfer and maternal transmission of Tc^r genes. Fecal fosmid libraries were functionally screened for Tc^r, and further PCR-screened for specific Tc^r genes. Tc^r fosmid inserts were sequenced at both ends to establish bacterial diversity. Mother and infant libraries contained Tc^r, although encoded by different genes and organisms. Tc^r organisms in the mother consisted mainly of Firmicutes and Bacteroidetes, and the main gene detected was tet(O), although tet(W) and tet(X) were also found. Identical Tc^r gene sequences were present in different bacterial families and even phyla, which may indicate horizontal transfer within the maternal GIT. In the infant library, Tc^r was present exclusively in streptococci carrying tet(M), tet(L) and erm(T) within a novel composite transposon, Tn6079. This transposon belongs to a family of broad host range conjugative elements, implying a potential for the joint spread of tetracycline and erythromycin resistance within the infant's gut. In addition, although not found in the infant metagenomic library, tet(O) and tet(W) could be detected in the uncloned DNA purified from the infant fecal sample. This is the first study to reveal the diversity of Tc^r bacteria in the human gut, to detect a likely transmission of antibiotic resistance from mother to infant GITs and to indicate the possible occurrence of gene transfers among distantly related bacteria coinhabiting the GIT of the same individual.