Rhodococcus equi is a soil-dwelling facultative intracellular pathogen that can
infect many mammals, including humans. R. equi is most well known for its
ability to cause severe pyogranulomatous disease in foals, primarily involving
the lungs although other body systems may also be affected. The disease is
endemic on many horse-breeding farms worldwide and poses a severe threat
to the horse breeding industry because there is no vaccine available. Current
prophylaxis is based on systematic preventative treatments with macrolides
combined with rifampicin, which are also used to treat clinical cases of the
disease in foals.
In this thesis I have used a combination of wet laboratory and
bioinformatic approaches to identify the molecular basis of emerging
combined resistance to macrolides and rifampicin in R. equi foal isolates
from the USA. The genomes of a selection of resistant and susceptible
strains from across the USA were sequenced and assembled. Resistance genes
were systematically searched by reciprocal best-match BLASTP comparisons
to known antibiotic resistance determinants. This led to the discovery
of a novel erythromycin ribosomal methylase (erm) gene, erm(46), in all
resistant strains. Complementation analysis in a susceptible R. equi strain showed that erm(46) was sufficient to confer resistance to all macrolides,
lincosamides, and streptogramin B. The erm(46) gene is carried by an
integrative conjugative element (ICE) which is transferable between R.
equi strains. The ICE is formed by two distinct parts, a class I integron
associated with an IS6100 sequence and the erm(46) determinant carried by a
sub-element which contains putative actinobacterial conjugative translocase
apparatus and a transposase/integrase. All resistant strains also carry
the same non synonymous point mutation in rpoB conferring rifampicin
resistance. Thus, these strains are carrying double resistance to the most
commonly used antibiotics to treat R. equi worldwide. Phylogenetic analysis
based on the core genome demonstrated that all resistant strains are clonal.
This indicates that although conjugal acquisition of the erm(46) conjugative
element may occur at a high frequency, the need for the concurrent presence
of a second rpoB mutation for survival in the macrolide and rifampicin
dominated farm environment has effectively selected for the spread of a single
clone.
In the second section of this work, we sequenced a further 20 R. equi
genomes from difference sources (equine, porcine, bovine, human), including
representatives of each of the seven major genogroups previously defined in
our laboratory based on pulsed field gel electrophoresis. I have used the
newly acquired genetic information to study the genome of R. equi and
analyse its diversity within and outwith its species group. This enabled
us to explore the pan genome and define that R. equi is a genetically
well-defined bacterial species. Our results provide definitive evidence that resolves the current dispute over R. equi classification, specifically they do
not support the recent proposal (based on classical polyphasic bacterial
taxonomical methods) that R. equi should be transferred to a new genus.
Our core-genome phylogenomic analyses unambiguously show that the genus
Rhodocococcus is monophyletic and that R. equi forms a clade together with
the most recently described related environmental species R. defluvii that
radiates from within the genus. Together with other shared biological and
genetic characteristics, namely the unique niche-adaptive mechanism based
on evolutionarily related extrachromosomal replicons, R. equi should be
conseidered a bona fide member of the genus Rhodococcus. We also confirm
that Rhodococcus spp. and Nocardia spp. are sufficiently distinct to warrant
them belonging to different genera.
In conclusion, this work used whole genome sequencing to characterize the
molecular basis underlying the emergence and clonal spread of multi-resistant
R. equi in horse breeding farms in the USA. This work also highlights the
limitations of classical taxonomical approaches in bacterial systematics, and
illustrates the importance of incorporating modern phylogenomic approaches
to understand the evolutionary relationships between bacterial strains and
their accurate taxonomic position