Genomic Insights into Drug Resistance and Virulence Platforms, CRISPR-Cas Systems and Phylogeny of Commensal E. coli from Wildlife

Abstract

Commensal bacteria act as important reservoirs of virulence and resistance genes. However, existing data are generally only focused on the analysis of human or human-related bacterial populations. There is a lack of genomic studies regarding commensal bacteria from hosts less exposed to antibiotics and other selective forces due to human activities, such as wildlife. In the present study, the genomes of thirty-eight E. coli strains from the gut of various wild animals were sequenced. The analysis of their accessory genome yielded a better understanding of the role of the mobilome on inter-bacterial dissemination of mosaic virulence and resistance plasmids. The study of the presence and composition of the CRISPR/Cas systems in E. coli from wild animals showed some viral and plasmid sequences among the spacers, as well as the relationship between CRISPR/Cas and E. coli phylogeny. Further, we constructed a single nucleotide polymorphisms-based core tree with E. coli strains from different sources (humans, livestock, food and extraintestinal environments). Bacteria from humans or highly human-influenced settings exhibit similar genetic patterns in CRISPR-Cas systems, plasmids or virulence/resistance genes-carrying modules. These observations, together with the absence of significant genetic changes in their core genome, suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems.

Keywords: CRISPR-Cas; E. coli; PLACNETw; WGS; antimicrobial resistance; wild animals.

Figure 1

Scheme of the CRISPR arrays located in CRISPR/Cas I-E and I-F1 systems found in the genome of our E. coli collection. Spacers are represented as vertical boxes and are oriented with respect to the leader (). Boxes with the same color represent spacers that appear in ≥2 strains. There were no overlapping spacer sequences between CRISPR 1, 2, 3 and 4 arrays, thus colors must be independently interpreted for each array. The different symbols indicate spacers matching at least 88% with phages (∆), plasmids (●) or IS (~)-like sequences in databases. Origin: deer (D); bird of prey (BP); rodent (R); wild boar (WB). CRISPR/cas I-E (A, A *, B, C) and I-F1 structures (A, B) as well as cas genes clusters (E1, E2, F, F*) correspond to those represented in Supplementary Materials Figures S1 and S2, respectively. ND: non-determined.

Figure 2

(a) Complete spacer sequences matching at least 97% with known plasmids or IS regions. (b) Distribution of CRISPR/Cas I-E and I-F1 systems among E. coli phylogroups.

Figure 3

Genetic environment and location of antimicrobial resistance determinants identified in E. coli from wildlife. (a) BLAST comparison of the novel resistance complex ∆Tn5393-bla_TEM-1a-strA-strB (MDR complex A) with other reference sequences from public databases; (b) BLAST comparison of a hybrid Tn21-1721 transposon carrying an atypical class 1 integron and tet(A) in non-conjugative IncR-IncFIA plasmids (MDR complex B). Genes are colored according to their function: purple, resistance to drug or metal; green, recombination/transposition; blue, replication and regulation of gene expression; grey, known function; white, unknown function.

Figure 4

PLACNETw reconstruction of the E. coli C7328 genome. After a manual pruning of the original network, four components were differentiated: the chromosome, plasmid 1 (small cryptic plasmid), plasmid 2 (IncN replicon) and plasmid 3 (IncR-IncFIA multi-replicon). Contigs are represented as blue nodes of size proportional to their length. Orange nodes indicate reference genomes. Contigs encoding plasmid relaxases and replication proteins are colored in red and yellow, respectively.

Figure 5

Distribution of the virulence determinants among E. coli phylogroups.

Figure 6

Genetic location and surrounding of iroN (a) and vat (b) virulence genes in some of the E. coli strains from our collection. The reference genomes used for BLASTn comparisons are indicated as “Ref. Genetic location-Strain name-(accession number)”. Genes are colored according to their function: orange, virulence genes; green, recombination/transposition; pink, prophage genes; grey, known function; white, unknown function.

Figure 7

Overview of two of the most populated branches of the core genome tree. The very low number of SNP differences observed between strains from wild animals and humans are highlighted in squares. Moreover, ST224 cluster also shows the higher discriminatory power of WGS when compared to conventional MLST typing.