Dissemination of IncI plasmid encoding bla CTX-M-1 is not hampered by its fitness cost in the pig's gut

Abstract

Multiresistance plasmids belonging to the IncI incompatibility group have become one of the most pervasive plasmid types in extended-spectrum beta-lactamase-producing Escherichia coli of animal origin. The extent of the burden imposed on the bacterial cell by these plasmids seems to modulate the emergence of "epidemic" plasmids. However, in vivo data in the natural environment of the strains are scarce. Here, we investigated the cost of a bla _CTX-M-1-IncI1 epidemic plasmid in a commensal E. coli animal strain, UB12-RC, before and after oral inoculation of 15 6- to 8-week- old specific-pathogen-free pigs. Growth rate in rich medium was determined on (i) UB12-RC and derivatives, with or without plasmid, in vivo and/or in vitro evolved, and (ii) strains that acquired the plasmid in the gut during the experiment. Although bla _CTX-M-1-IncI1 plasmid imposed no measurable burden on the recipient strain after conjugation and during the longitudinal carriage in the pig's gut, we observed a significant difference in the bacterial growth rate between IncI1 plasmid-carrying and plasmid-free isolates collected during in vivo carriage. Only a few mutations on the chromosome of the UB12-RC derivatives were detected by whole-genome sequencing. RNA-Seq analysis of a selected set of these strains showed that transcriptional responses to the bla _CTX-M-1-IncI1 acquisition were limited, affecting metabolism, stress response, and motility functions. Our data suggest that the effect of IncI plasmid on host cells is limited, fitness cost being insufficient to act as a barrier to IncI plasmid spread among natural population of E. coli in the gut niche.

Keywords: CTX-M; ESBL; IncI plasmid; RNA-Seq; fitness cost.

Fig 1

Boxplot representation of the maximum growth rate (MGR) of each pool lineage: MGR of the donor strain (M63-DN), the recipient strain (UB12-RC), the ancestral transconjugant (UB12-TC, IncI, pST3), seven in vivo evolved transconjugants (UB12-TC^ive), and three evolved transconjugants which had lost in vivo the IncI plasmid (UB12-TCΔpIncI). UB12-TC^ive and UB12-TCΔpIncI were isolated in stools between day 7 and day 45 during longitudinal study of in vivo carriage in pigs. For each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Each point corresponds to one measurement per clone, which were repeated six times per strains. Asterisks indicate significant differences (* =P < 0.05, ** =P < 0.01 and *** =P < 0.001). NS = not significant.

Fig 2

Boxplot representation of the maximum growth rate (MGR) of each pool lineage: MGR of five ancestral UB12-RC and UB12-TC control strains and evolved UB12-RC and UB12-TC strains at day 30. For each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Each point corresponds to one measurement per clone, which were repeated six times per transconjugant and control. NS = not significant.

Fig 3

(A) Phylogenetic tree of the 11 newly formed transconjugants detected among pig E. coli commensal isolates (NI clones, E. coli, IncI, pST3), the M63-DN donor strain, and the UB12-TC transconjugant strain (in bold) are presented. The tree is rooted on an Escherichia clade (E1492cladeI (27)). The phylogenetic tree was constructed with IQ-TREE v1.6.9 from the core genome genes using Roary v1.007002. From left to right are presented the Sequence Type (according to the Warwick University scheme), the O and H serotypes, the fimH allele, and the fitness cost of NI clones compared to the donor strain (M63-DN) (NA = not applicable, NS = not significant, * =P < 0.05, ** =P < 0.01, and *** =P < 0.001). Each branch of the same phylogenetic group is colored (yellow, D; red, B2; orange, G; blue, A; light green, C; dark green, B1). Information on O group, H type, and fimH allele are given in Table 1. (B) Boxplot representation of the maximum growth rate (MGR) of each strain : donor strain (M63-DN), UB12-TC ancestral transconjugant and NI clones, isolated in stools between day 4 and day 22 during longitudinal study of in vivo carriage in pigs. For each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. Each point corresponds to one measurement per clone, which were repeated six times per strains. Asterisks in red indicate significant differences (* =P < 0.05, ** =P < 0.01, and *** =P < 0.001) in comparison to M63-DN. Colors correspond to the strains of the same phylogroup (color code as in A).