Culture-Independent Genome Sequencing of Clinical Samples Reveals an Unexpected Heterogeneity of Infections by Chlamydia pecorum

Abstract

bChlamydia pecorum is an important global pathogen of livestock, and it is also a significant threat to the long-term survival of Australia's koala populations. This study employed a culture-independent DNA capture approach to sequence C. pecorum genomes directly from clinical swab samples collected from koalas with chlamydial disease as well as from sheep with arthritis and conjunctivitis. Investigations into single-nucleotide polymorphisms within each of the swab samples revealed that a portion of the reads in each sample belonged to separate C. pecorum strains, suggesting that all of the clinical samples analyzed contained mixed populations of genetically distinct C. pecorum isolates. This observation was independent of the anatomical site sampled and the host species. Using the genomes of strains identified in each of these samples, whole-genome phylogenetic analysis revealed that a clade containing a bovine and a koala isolate is distinct from other clades comprised of livestock or koala C. pecorum strains. Providing additional evidence to support exposure of koalas to Australian livestock strains, two minor strains assembled from the koala swab samples clustered with livestock strains rather than koala strains. Culture-independent probebased genome capture and sequencing of clinical samples provides the strongest evidence yet to suggest that naturally occurring chlamydial infections are comprised of multiple genetically distinct strains. Chlamydia pecorum is a widespread pathogen of economically important livestock species, such as cattle and sheep. In cattle, C. pecorum is associated with sporadic bovine encephalomyelitis (SBE), which presents as a fever followed by limb stiffness and staggering (1). In sheep, C. pecorum infections commonly are linked to polyarthritis and conjunctivitis, which can spread rapidly in a flock (2, 3). While these infections are economically relevant to producers, most C. pecorum infections in ruminants appear to be asymptomatic or subclinical, characterized by a consistent presence in the gastrointestinal tract (4, 5). While questions remain over the impact of these infections in livestock globally, the best example of the pathogenic potential of this obligate intracellular bacterium actually is found in koalas, a native Australian marsupial that continues to experience localized extinctions. C. pecorum infections in koalas can cause debilitating ocular and urogenital tract diseases (6, 7). Epidemiological questions have been raised about the relationships between C. pecorum strains infecting domesticated animals and the koala, with a recent C. pecorum multilocus sequence typing (MLST) study revealing the presence of identical sequence types in samples collected from each host (8). As a follow-up to these studies, we recently sequenced the genomes of several cultured koala C. pecorum isolates, revealing a high degree of synteny and sequence identity (98.5 to 98.8%) with C. pecorum genomes from European and U.S. cattle and sheep (9).High-throughput comparative genome sequencin...

Chlamydia pecorum: successful pathogen of koalas or Australian livestock?

“…pCpec seems to be highly distributed in strains, however plasmidless strains are also common, raising questions over its function in disease pathogenesis 26 . Culture-independent genome sequencing of sheep, cattle and koala C. pecorum positive clinical swabs demonstrated that not only could multiple strains infect a single host but that a single anatomical site can harbour multiple C. pecorum strains 27 , somewhat confirming observations from molecular epidemiology but also reiterating complex epidemiology of these infections.…”

“…pecorum genome-derived phylogenies highlighted the polyphyletic history of the pathogen itself, and provided important clues about the evolutionary origins of koala strains, giving more confidence to our hypothesis that the origin of C. pecorum infections in Australia is associated with importation of domesticated animals into Australia with European colonisation 3,25,27 .…”

Culture-independent genomic characterisation of Candidatus Chlamydia sanzinia, a novel uncultivated bacterium infecting snakes

“…While our group and others have recently used several genome sequencing methods to broaden our knowledge of previously described species in the genus Chlamydia [13–17], the current study suggests that a shotgun deep sequencing approach is better suited to novel species. For instance, using “bait” probes designed from a reference genome risks overlooking previously undetected or undescribed features, such as a plasmid [25].…”

HapFlow: Visualising haplotypes in sequencing data

“…C. pecorum DNA was extracted directly from the host 82 cell contaminants using Sure-Select RNA probes and sequenced using an Illumina Hi-Seq to 83 produce 101bp paired-end reads, as previously described (Bachmann, et al, 2015). These 84 reads were then mapped back to E58 using Bowtie-2 and then variant calling was performed 85 using FreeBayes.…”

“…Infections frequently contain multiple strains 24 of the same species (Darch, et al, 2015;Taylor, et al, 1995). This has important implications 25 for detecting transmission events (Bachmann, et al, 2015) and determining treatment 26 outcomes (Cohen, et al, 2012). Several methods have been developed to analyse mixed-27 strain populations.…”