The distribution of fitness effects of plasmid pOXA-48 in clinical enterobacteria

Abstract

Antimicrobial resistance (AMR) in bacteria is a major public health problem. The main route for AMR acquisition in clinically important bacteria is the horizontal transfer of plasmids carrying resistance genes. AMR plasmids allow bacteria to survive antibiotics, but they also entail physiological alterations in the host cell. Multiple studies over the last few years have indicated that these alterations can translate into a fitness cost when antibiotics are absent. However, due to technical limitations, most of these studies are based on analysing new associations between plasmids and bacteria generated in vitro, and we know very little about the effects of plasmids in their native bacterial hosts. In this study, we used a CRISPR-Cas9-tool to selectively cure plasmids from clinical enterobacteria to overcome this limitation. Using this approach, we were able to study the fitness effects of the carbapenem resistance plasmid pOXA-48 in 35 pOXA-48-carrying isolates recovered from hospitalized patients. Our results revealed that pOXA-48 produces variable effects across the collection of wild-type enterobacterial strains naturally carrying the plasmid, ranging from fitness costs to fitness benefits. Importantly, the plasmid was only associated with a significant fitness reduction in four out of 35 clones, and produced no significant changes in fitness in the great majority of isolates. Our results suggest that plasmids produce neutral fitness effects in most native bacterial hosts, helping to explain the great prevalence of plasmids in natural microbial communities.

Keywords: DFE; Plasmid cost; antimicrobial resistance; carbapenemase; conjugation; enterobacteria; pOXA-48; plasmids.

Fig. 1.

Bacterial collection used in this study. Summary of pOXA-48-carrying strains present in the entire collection and the subset used for this study. (a) Distribution of multilocus sequence types (STs) for E. coli , Klebsiella pneumoniae and other species in the collection of wild-type pOXA-48-carrying enterobacteria. (b) Representative subsample of the collection selected for this study. (c) Strains successfully cured from pOXA-48. Colours indicate different STs (upper and central rows) or different species (lower row). The most representative STs in E. coli and K. pneumoniae are indicated. Kva: Klebsiella variicola ; Cfr: Citrobacter freundii ; Kas: Kluyvera ascorbata . (d) Unrooted phylogeny reconstructed with the genomic sequences of the 35 strains cured from pOXA-48. Branch length represents mash distances between whole-genome assemblies. Scale breaks were introduced for long branches. The STs are also indicated.

Fig. 2.

Experimental design. A representative subsample of wild-type pOXA-48-carrying enterobacteria (n=60) was selected from a collection isolated from the gut microbiota of hospitalized patients (n=225). Plasmid pOXA-48 was eliminated (cured) from the wild-type strains using a CRISPR-Cas9-based method (see Methods). Growth curves and competition experiments were performed for all the pOXA-48-carrying and pOXA-48-free clones. We used the relative fitness data obtained from the competition assays to determine the distribution of fitness effects (DFE) of pOXA-48 in its natural hosts.

Fig. 3.

Fitness effects of plasmid pOXA-48 in wild-type clinical enterobacteria. (a) Relative fitness (w) of pOXA-48-carrying clones compared to their corresponding pOXA-48-free isogenic versions (see Methods). Values >1 indicate that plasmid pOXA-48 is associated with a fitness advantage, while values <1 indicate a fitness reduction associated with pOXA-48 carriage. Bars represent normalized relative fitness (average of six independent biological replicates of the competition pOXA-48-carrying vs. E. coli J53 divided by the average of six independent biological replicates of the competition pOXA-48-free vs. E. coli J53). Error bars represent the propagated standard error. Asterisks indicate significant costs or benefits associated with pOXA-48 carriage (Bonferroni-corrected paired t tests, P<0.05). Strain AJ_N46 is an outlier (Grubbs test, P=1.07×10⁻⁶). (b) Comparison of pOXA-48 DFE in the collection of naive, ecologically compatible pOXA-48 carriers [17] and in the collection of pOXA-48 natural carriers analysed in this work. Bars indicate the number of strains in each relative fitness bin. Dashed lines indicate the mean relative fitness of each collection. Both distributions are normal when not including the outlier AJ_N46 (Shapiro–Wilk normality tests: [17], P=0.14; this work, P=0.095). Cumulative distribution functions for both collections are represented in Fig. S4.