Within-patient evolution of plasmid-mediated antimicrobial resistance

Abstract

Antimicrobial resistance (AMR) in bacteria is a major threat to public health; one of the key elements in the spread and evolution of AMR in clinical pathogens is the transfer of conjugative plasmids. The drivers of AMR evolution have been studied extensively in vitro but the evolution of plasmid-mediated AMR in vivo remains poorly explored. Here, we tracked the evolution of the clinically relevant plasmid pOXA-48, which confers resistance to the last-resort antibiotics carbapenems, in a large collection of enterobacterial clones isolated from the gut of hospitalized patients. Combining genomic and experimental approaches, we first characterized plasmid diversity and the genotypic and phenotypic effects of multiple plasmid mutations on a common genetic background. Second, using cutting-edge genomic editing in wild-type multidrug-resistant enterobacteria, we dissected three cases of within-patient plasmid-mediated AMR evolution. Our results revealed compensatory evolution of plasmid-associated fitness cost and the evolution of enhanced plasmid-mediated AMR in bacteria evolving in the gut of hospitalized patients. Crucially, we observed that the evolution of pOXA-48-mediated AMR in vivo involves a pivotal trade-off between resistance levels and bacterial fitness. This study highlights the need to develop new evolution-informed approaches to tackle plasmid-mediated AMR dissemination.

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Figure 1. pOXA-48 plasmid variants tested in E. coli J53.

A) Representation of pOXA-48 variants (PVs) A-N in concentric circles (for variant details see legend). The gene map in the outer circle represents the most common variant (PV-I, used as a reference and highlighted by grey shading in panels B-E). Arrows indicate the reading frames, and colours indicate gene functional classification. Gene names are indicated in the outer circle, and the names of genes showing mutations are represented inside boxes. B) Box plots showing relative fitness (w) of pOXA-48-carrying E. coli J53 relative to plasmid-free J53. Horizontal lines inside boxes indicate median values, the upper and lower hinges correspond to the 25^th and 75^th percentiles, and whiskers extend to observations within the 1.5 x the interquartile range (IQR). Individual points show independent replicates (n=6). C) Resistance to ertapenem (ERT) in plasmid-free and plasmid-carrying E. coli J53, represented as the 90% inhibitory concentration (IC90) in mg/L. Bars indicate the mean values and dots indicate individual replicates (n=10 for PV-K & PV-E and n=5 for the remaining PVs). Black bars represent the standard error of the mean. D) Conjugation rates of different PVs in E. coli J53 (in log₁₀ scale, n=14 for PV-I, n=9 for PV-J, n=8 for PV-E & PV-K, n=3 for PV-A, and n=6 for the remaining PVs), represented as boxplots as in B. E) Plasmid copy number of different PVs in E. coli J53, represented as boxplots as in B (n=6). Asterisks in panels B-E indicate significance for the comparison of each PV with PV-I (two-sided pairwise comparison Wilcoxon rank-sum exact test with FDR correction P<0.01 after two-sided Kruskal-Wallis test). F) Correlation between relative fitness (w) and resistance to ertapenem (mean IC90) in PV-carrying E. coli J53. Individual points indicate the mean value, and lines represent the standard error of the mean IC90 and the propagated standard error of the relative fitness. The size of each point is proportional to PCN in J53. The diamond represents the plasmid-free J53 values, which were not included in the correlation. Individual PVs are indicated by letters. The red dashed line indicates the regression and the gray-shaded zone covers the 95% confidence interval.

Figure 2. Screening the within-patient evolution of pOXA-48-mediated AMR.

A) Timelines of the isolation of pOXA-48-carrying enterobacteria from patients HKH, JWC, and WDV (see legend). Isolate features are detailed in the legend. Swab dates are indicated next to the timeline (day/month). pOXA-48-selecting antibiotic treatments are indicated in the timeline (MER, meropenem; ERT, ertapenem; AMC, amoxicillin + clavulanic acid). Each PVs is indicated by a letter. Species are indicated by letters and symbols (KPN and circle for K. pneumoniae; EC and square for E. coli). The multilocus sequence-type code is indicated next to the species label. Isolates in which the new PV was detected are indicated by blue type and larger size. The patient JWC timeline reveals the emergence of two K. pneumoniae isolates co-isolated on an agar plate supplemented with ertapenem 0.3 mg/L. B-D) Genetic relationship built using core-genome comparisons (midpoint rooted phylogenetic trees) of K. pneumoniae ST11 (n=85, B), K. pneumoniae ST307 (n=20, C), and E. coli ST10 and ST744 (n=12 & n=2 respectively, D) from the collection. Strain designation, patient codes, and PVs are indicated (see Supplementary Data 1). Isolates involved in putative cases of within-patient evolution are highlighted in blue. Bold lettering marks the isolate in which the novel variant was identified. The tree-scale indicates nucleotide substitution per site.

Figure 3. Characterization of the in vivo evolution of plasmid-mediated AMR.

A) Workflow used to investigate the within-patient evolution of pOXA-48-mediated AMR. B) Relative fitness (w) of each plasmid-bacteria combination compared with the plasmid-free strain (see Methods). Horizontal lines inside boxes indicate median values, the upper and lower hinges correspond to the 25th and 75th percentiles, and whiskers extend to observations within 1.5 × the IQR. Individual points represent independent replicates (n=18). Asterisks in panels B-D indicate significant differences (P<0.05 in pairwise comparison two-sided Wilcoxon rank-sum exact test with FDR correction in B and C, and Padj<0.05 by one-way ANOVA in D); ns, nonsignificant (Padj>0.05). C) Resistance to ertapenem (ERT) measured as IC90 (mg/L) of plasmid-free and plasmid-carrying combinations. Lines indicate median values and individual replicates are indicated by points (n=5). D) Plasmid copy number (PCN) of each PV, represented in boxplots as in B (n=6). E) Schematic representations of the trade-off between antibiotic resistance (median IC90) and relative fitness (median w) in the strains under study. Black arrows represent the trade-off and arrowheads indicate the PVs timeline (from ancestral to novel PV).