Use of single molecule sequencing for comparative genomics of an environmental and a clinical isolate of Clostridium difficile ribotype 078

Abstract

BackgroundHow the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome).ResultsChromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM) and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for m4C methylation, but has one additional spacer when compared to the clinical isolate M120.ConclusionsThese findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3346-2) contains supplementary material, which is available to authorized users.

Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance

“…We found 30% of ST11 isolates had a TetR phenotype conferred by efflux and ribosomal protective proteins, expressed by Tn 6190 ( tetM + ) and Tn 6164 ( tet-44 + ). Tn 6190 has a strong affiliation with the RT126/078 lineages, present in 79.7% and 55.0% of strains in this study, respectively, and to date only reported in these RTs (14, 16, 41). Similarly, Tn 6164 has been found in RT078 only and, prior to this study, only within Europe.…”

“…However, neonatal calves and piglets in Australia are reservoirs for a number of other ST11 RTs, notably 126, 127, 033, and 288 (11–13), all of which have been isolated from humans with CDI in Australia, Europe, the Middle East, and Asia (1, 2). While there has been a focus on the genome and evolution of RT078 (4, 14–16), no such studies have been performed on other ST11 RT sublineages.…”

Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis

“…Clostridioides (formerly Clostridium) difficile is a spore-forming Gram-positive obligate anaerobe and the leading cause of nosocomial antibiotic-associated disease in the developed world 1 (Supplementary Notes). Despite the significant progress achieved in the understanding of C. difficile physiology, genetics, and genomic evolution 2,3 , the roles played by epigenetic factors, namely DNA methylation, have not been systematically studied [4][5][6] . In the bacterial kingdom, there are three major forms of DNA methylation: N6-methyladenine (6mA, the most prevalent form representing ~80%), N4-methylcytosine (4mC), and 5methylcytosine (5mC).…”

Clostridium difficile infection: Evolution, phylogeny and molecular epidemiology

“…It can still be improved with a more robust molecular clock, as estimates of mutation rates are needed for each lineage and different hosts. Whole genome sequencing will only improve with the introduction of long read sequencing chemistries such as the Nanopore and PacBio (Hargreaves et al, 2016).…”