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Phylogenetic relationship of Ornithobacterium rhinotracheale strains

The bacterium Ornithobacterium rhinotracheale is associated with respiratory disease in wild birds and poultry. In this study, the phylogenetic analysis of nine reference strains of O. rhinotracheale belonging to serovars A to I, and eight Mexican isolates belonging to serovar A, was performed. The analysis was extended to include sequences from another 23 strains available in the public domain. The analysis showed that the 40 sequences formed six clusters, I to VI. All eight Mexican field isolates were placed in cluster I. One of the reference strains appears to present genetic diversity not previously recognized and was placed in a new genetic cluster. In conclusion, the phylogenetic analysis of O. rhinotracheale strains, based on the 16S rRNA gene, is a suitable tool for epidemiologic studies.

by construction of a multiple alignment by ClustalX, removal of gapped columns, and analysis by the maximum likelihood method [16] and genetic distances obtained by Kimura's 2-parameter model, conducted using MEGA5 [6]. Additional sequences of well-characterized reference strains and isolates from GenBank and Green Genes data-bases were included in the analysis (Fig. 1) [2,18,19]. Shorter sequences (less than 700 nucleotides) were not included in the analysis [4,8].
Sequences for the 16S rRNA gene of the nine reference strains and eight Mexican isolates of O. rhinotracheale were obtained and deposited in GenBank (accession numbers KY612254 and KY809786 to KY809801). Pairwise sequence comparisons revealed 99.1 to 100% sequence identity among the 40 sequences included in the study. The analysis of polymorphic nucleotides in the 16S rRNA gene showed differences from 1 to 10 nucleotides in comparison to a consensus 16S rRNA gene sequence of O. rhinotracheale ( Table 2). The 40 sequences included into the phylogenetic analysis were clustered into six genetic clusters (I to VI; Fig. 1).
As the study of Amonsin et al. [2] had identified a system for correlating the ET typing results with single nucleotide polymorphisms (SNP) in the 16S rRNA gene, the SNP patterns of all of the strains and field isolates in this study were examined using this correlation ( Table 2). Some of the sequences used in the current study were the same as used in the Amonsin et al. [2] study. Hence, these strains and isolates have a known ET assignment. For a number of other strains and isolates, the correlation proposed by Amonsin et al. [2] was used to predict the ET (predicted ET is marked by an asterisk in Table 2). Some of the strains and isolates had the SNP pattern noted by Amonsin et al. [2] as well as additional SNPs not noted by Amonsin et al. [2]. Hence, for these strains and isolates, the predicted ET is marked as a variant ( Table 2). The final group of strains and isolates had SNP profiles that were markedly different from those recorded by Amonsin et al. [2] and it was not possible to predict an ET ( Table 2).
All of the strains previously typed as ET 1 and ET 2 by Amonsin et al. [2] were assigned to cluster I in the current study ( Fig. 1 and Table 2). As 17 of the 19 strains and isolates allocated to cluster I aligned with the scheme suggested by Amonsin et al. [2] for correlating polymorphisms in the 16S rRNA gene sequence, we were able to predict that these 13 strains/isolates would be ET 1 ( Table 2). A further six strains/isolates within cluster I had the same SNP pattern as assigned by Amonsin et al. [2] to ET 1 but also had additional SNP sites not noted by Amonsin et al. [2] and were thus predicted to be variants of ET 1 (termed ET 1v in Table 2). Cluster II consisted of strain LMG-11553 (GenBank accession number U87102). Cluster III consisted of strain LMG-15870 (GenBank accession number U87103) of known ET 5 and four strains that could not be assigned to a predicted ET. Cluster IV consisted of strain LMG-11554 (GenBank accession number U87104) of known ET 6 and one isolate that could not be assigned to a predicted ET. Cluster V consisted of strain LMG-14578 (GenBank accession number U87105) of known ET 4 and three reference strains that were assigned to a predicted ET. Cluster VI had only a single reference strain that could not be assigned to an ET.
In this study, we report the sequencing of 16S rRNA gene from nine reference strains of Ornithobacterium rhinotracheale belonging to serovars A to I. While the sequencing of 16S rRNA gene from eight reference strains of O. rhinotracheale belonging to serovars A to H has been previously reported [9], the sequences are not available in public data-bases.
Amonsin et al. [2] assigned 90.9% (50 of 55) of isolates of O. rhinotracheale to ET 1 and ET 2, comprising the ET 1 complex. In the present study, phylogenetic analysis of 16S rRNA gene sequences resulted in a cluster (cluster I) that consisted predominantly of strains and isolates that were known to be ET 1 or ET 2 or predicted to be ET 1 or a variant of ET 1, confirming the ET 1 complex. All of the Mexican serovar A isolates and reference strains of serovars A, B, E and G were clustered in cluster I (Fig. 1). Clearly, this complex (16S rRNA gene cluster I/ET complex 1) is a cluster of strains and isolates that are commonly distributed around the world. In the current study, more variation in the 16S rRNA gene was found than was used by the earlier study of Amonsin et al. [2] to correlate ET with 16S rRNA sequences. Despite this increased diversity, there was still a good correlation between ET results (predicted or known) and the phylogeny.
Reference strain ORV 94084 K858 of serovar F of O. rhinotracheale was allocated to cluster VI as the sole member of that cluster. Ten nucleotide changes were recorded in this strain ( Table 2). It is clear that this strain represents significant new diversity that has not been previously recognized and association with antigenic and pathogenic traits need to be studied. This reference strain was included in a multilocus sequence typing (MLST) analysis that included seven housekeeping genes, and showed the most nucleotide polymorphisms [15,16]. Two distinct phylogenetic clusters were identified in the phylogenetic tree generated from MLST sequences and reference strain ORV 94084 K858 of serovar F of O. rhinotracheale was allocated, along with other two strains, to cluster A and differed considerably from the vast majority of O. rhinotracheale strains included in that study [16]. Further studies comparing phylogenetic analysis based on 16S rRNA gene and MLST are needed.
Identical genotypes (16S rRNA gene phylogeny and ERIC-PCR pattern) of Mexican field isolates included in the study, recovered over a long time period in two main Mexican geographic areas (Puebla and Jalisco), support that O. rhinotracheale population is predominantly clonal as has been previously suggested [2,19].
In conclusion, new information was added to the knowledge of population structure of O. rhinotracheale by the phylogenetic analysis of the 16S rRNA gene, allowing a worldwide comparison of sequence data and evidencing the existence of greater genetic variability among strains than previously contemplated.
ACKNOWLEDGMENTS. This work was partially funded by Universidad Autónoma del Estado de México, project 212866, and Solutions in Immunology and Microbiology (SIM) S.A. de C.V., project 2016-1. a) Strains with underlining were examined in the study of Amonsin et al. [2]. The polymorphisms reported for these strains are based on the Genbank sequence. b) ET=Electrophoretic type. The results that are underlined are those reported by Amonsin et al. [2]. The ET results with an asterisk (*) are predicted results based on the current sequencing data and an exact alignment with the polymorphism pattern reported by Amonsin et al. [2]. Where additional polymorphisms were found outside the base positions used by Amonsin et al. [2], the ET is a predicted variant ET (1v). Where the polymorphisms in the strains do not match at all with the polymorphisms used by Amonsin et al. [2], the result is recorded as ? c) ERIC-PCR genotype=the enterobacterial repetitive intergenic consensus-type as previously defined [10]. d) Cluster assignment is based on Fig. 1