Whilst searching for a molecular method to identify the different species of Raoultella and Klebsiella oxytoca, it was observed that the OXY-1 and OXY-2 β-lactamase-producing K. oxytoca isolates displayed two distinguishable enterobacterial repetitive intergenic consensus (ERIC)-1R profiles. It was hypothesized that the two groups of chromosomal β-lactamases might correspond to two groups of strains in the K. oxytoca taxon. To confirm this hypothesis, clinical isolates and reference strains of K. oxytoca were studied by determination of the sequence of their bla OXY genes, and of a partial fragment of their 16S rRNA (387 bp) and rpoB (512 bp) genes. The sequence data were phylogenetically analysed by using the parsimony method. Four clinical isolates possessed a bla OXY-1 gene and nine possessed a bla OXY-2 gene. The mean percentage of rpoB and 16S rRNA gene identity was >99 % within each group of strains, whereas it was 96·56±0·24 % for rpoB genes and 97·80±0·22 % for 16S rRNA genes between the group of strains harbouring the bla OXY-1 gene and the group harbouring the bla OXY-2 gene. The phylogenetic tree resulting from combined analysis of the 16S rRNA and rpoB datasets showed that the K. oxytoca isolates were monophyletic and separated into two clades; these clades included strains with either the bla OXY-1 gene or the bla OXY-2 gene. This result was supported with high bootstrap values of 97 and 99 %, respectively. Moreover, the two groups of strains displayed distinct ERIC-1R profiles, with bands characteristic of each profile. Thus, the chromosomal bla OXY gene sequence is able to delineate not only two groups of β-lactamases in K. oxytoca, but also two clades in the K. oxytoca taxon, in a manner similar to the sequence of housekeeping genes. These results suggest that K. oxytoca should be divided into two genetic groups, group OXY-1 represented by K. oxytoca strain SL781 (=CIP 104963) and group OXY-2 by K. oxytoca strain SL911 (=CIP 106098).
The nucleotide sequences of the 3′ end of the 16S rDNA and the 16S–23S internal transcribed spacer (ITS) of 40 Bacillaceae species were determined. These included 21 Bacillus, 9 Paenibacillus, 6 Brevibacillus, 2 Geobacillus, 1 Marinibacillus and 1 Virgibacillus species. Comparative sequence analysis of a 220 bp region covering a highly conserved 150 bp sequence located at the 3′ end of the 16S rRNA coding region and a conserved 70 bp sequence located at the 5′ end of the 16S–23S ITS of the 40 species and six sequences available in GenBank were used to infer the phylogenetic relationships between all 46 taxa. When a maximal distance (D max, where D refers to the number of nucleotide substitutions per site) of 0·31 was introduced as a threshold to determine groupings, 10 phylogenetically distinct clusters were revealed. Twenty-six Bacillus species were separated in seven groups (I, II, III, IV, V, VI and X), but Bacillus circulans remained ungrouped. All six Brevibacillus species under study were in Group VII. The nine Paenibacillus species fell into two distinct groups (VIII and IX). Species with D max values within 0·05 were considered to be very closely related. These were Bacillus psychrophilus and Bacillus psychrosaccharolyticus in Group II; ‘Bacillus maroccanus’ and Bacillus simplex in Group II; Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus mojavensis and Bacillus subtilis in Group VI; Bacillus fusiformis and Bacillus sphaericus in Group VI; Brevibacillus brevis and Brevibacillus formosus in Group VII; Paenibacillus gordonae and Paenibacillus validus in Group VIII; and Bacillus anthracis, Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis in Group X. The phylogenetic classification presented here is, in general, in agreement with current classifications based on phenotypic and molecular data. Our findings suggest, however, that in some cases, further divisions or, conversely, further groupings might be warranted. Should current classifications be re-examined in the light of our results, D max values of 0·31 and 0·05, as exemplified here, may prove useful threshold values for the grouping of Bacillaceae into taxa akin to genera and species, respectively. These D max thresholds may also reveal, in a different way, bacterial species for which further characterization might be warranted for proper classification and/or reassignment.
The present study was aimed at simplifying procedures to delineate species and identify isolates based on DNA–DNA reassociation. DNA macro-arrays harbouring genomic DNA of reference strains of several Burkholderia species were produced. Labelled genomic DNA, hybridized to such an array, allowed multiple relative pairwise comparisons. Based on the relative DNA–DNA relatedness values, a complete data matrix was constructed and the ability of the method to discriminate strains belonging to different species was assessed. This simple approach led successfully to the discrimination of Burkholderia mallei from Burkholderia pseudomallei, but also discriminated Burkholderia cepacia genomovars I and III, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis and Burkholderia vietnamiensis. Present data showed a sufficient degree of congruence with previous DNA–DNA reassociation techniques. As part of a polyphasic taxonomic scheme, this straightforward approach is proposed to improve species definition, especially for application in the rapid screening necessary for large numbers of clinical or environmental isolates.
Pathological and serological evidence and DNA–DNA reassociation data indicate that Chlamydophila psittaci and Chlamydophila abortus are separate species. C. psittaci causes avian systemic disease and C. abortus causes abortion. Both previously belonged to Chlamydia psittaci are associated with zoonotic and enzootic outbreaks. Genetic studies suggest that they are closely related and because of the recent availability of diverse C. psittaci strains and comparative data for several genes, it was possible to explore this relationship. The parrot C. psittaci strain 84/2334 was found to have DNA sequences that were identical to an extrachromosomal plasmid in duck C. psittaci strain N352, to rnpB in strain R54 from a brown skua and to the rrn intergenic spacer in parakeet strain Prk/Daruma (from Germany, Antarctica and Japan, respectively). Analysis of ompA and the rrn spacer revealed progressive diversification of the strains, with 84/2334 resembling what might have been a recent ancestor of C. abortus. Another C. psittaci strain (VS225) showed evidence of having undergone convergent evolution towards the C. abortus-like genotype, whereas strain R54 diverged independently. For the first time, these studies link C. abortus in an evolutionary context to the C. psittaci lineage. It has been concluded that C. abortus diverged from C. psittaci, and so strain R54 was designated a C. psittaci strain. It is recommended that characterization of C. psittaci and C. abortus strains should utilize more than a single method and more than a single gene.
The phylogenetic relationships of all known species of the genus Aeromonas were investigated by using the sequence of gyrB, a gene that encodes the B-subunit of DNA gyrase. Nucleotide sequences of gyrB were determined from 53 Aeromonas strains, including some new isolates, which were also characterized by analysis of the 16S rDNA variable regions. The results support the recognition of the family Aeromonadaceae, as distinct from Plesiomonas shigelloides and other enteric bacteria. This phylogenetic marker revealed strain groupings that are consistent with the taxonomic organization of all Aeromonas species described to date. In particular, gyrB results agreed with 16S rDNA analysis; moreover, the former showed a higher capacity to differentiate between species. The present analysis was useful for the elucidation of reported discrepancies between different DNA–DNA hybridization sets. Additionally, due to the sequence diversity found at the intraspecies level, gyrB is proposed as a useful target for simultaneous identification of species and strains. In conclusion, the gyrB gene has proved to be an excellent molecular chronometer for phylogenetic studies of the genus Aeromonas.