Molecular analysis of the chromosomal 16S rRNA gene and vapA plasmid gene of Polish field strains of R. equi

Abstract

Rhodococcus equi (R. hoagii) is an opportunistic pathogen commonly found in foals up to 6 months old and animal environment. The R. equi genome contains genetically stable chromosomal DNA and an 80-90 kb plasmid containing vapA gene, responsible for virulence. Most reports from around the world focus on the determination of R. equi plasmid profiles. Few studies have attempted to determine differences in nucleotide sequences between virulent strains of R. equi isolated from foals and breeding environment. The aim of the study was to perform a molecular analysis of a fragment of the chromosomal gene encoding the 16S rRNA subunit and the vapA plasmid gene of virulent R. equi strains isolated on Polish studs from foals and from the breeding environment of horses. The sequencing method was used to compare the primary structure of fragments of the chromosomal and plasmid DNA of the virulent R. equi strains. The sequences of 22 clinical and 18 environmental R. equi isolates were compared with the sequences of the gene fragments of reference strains available in the NCBI GenBank database. All sequenced 16S rRNA amplicons of Polish field strains were identical and showed 99.5% similarity to the four randomly selected sequences of this gene fragment in the GenBank database. The results confirm that fragments of the 16S rRNA gene of R. equi strains are highly conserved and do not undergo variation in field conditions. Analysis of the sequencing results for the vapA gene fragment of the strains used in our study revealed two polymorphic variants and clear differences between the sequences of strains isolated from foals and from soil samples. Presumably, R. equi strains present in the breeding environment are more exposed than clinical strains to adverse external factors. This may result in changes in the DNA sequence due to natural selection.

Fig 1. Location of stud farms (A-K) selected for the study.

Fig 2. Dendrogram of the similarity of nucleotide sequences of a fragment of the gene (441 bp) encoding the 16S rRNA subunit of R. equi strains.

DSM 20307, pony1, DSM 777, BJ13 –sequences of R. equi strains from the NCBI GenBank database; 1–22 –clinical strains of R. equi; 23–40 –environmental strains of R. equi.

Fig 3. Percentage of identity between sequences of a fragment of the gene encoding the 16S rRNA subunit of R. equi strains.

1–22 –clinical strains of R. equi; 23–40 –environmental strains of R. equi; BJ13, DSM 777, DSM 20307, pony1 –sequences of strains from the NCBI GenBank database.

Fig 4. Dendrogram of the similarity of nucleotide sequences of a fragment of the vapA gene (875 bp) of R. equi strains.

1–21 –clinical strains of R. equi with the 85-kb type I plasmid profile; 22 –clinical strain of R. equi with the 87-kb type I plasmid profile; ATCC 33701, pVAPA1037 –sequences of R. equi reference strains from the NCBI GenBank database; 23–30, 31–36, 37–40 –environmental strains of R. equi.

Fig 5. Percentage of identity between sequences of a fragment of the vapA gene of R. equi strains.

1–21 –clinical strains of R. equi with the 85-kb type I plasmid profile; 22 –clinical strain of R. equi with the 87-kb type I plasmid profile; 23–30, 31–36, 37–40 –environmental strains of R. equi; ATCC 33701, pVAPA1037 –sequences of R. equi reference strains from the NCBI GenBank database.