A Broad-Host-Range Plasmid Outbreak: Dynamics of IncL/M Plasmids Transferring Carbapenemase Genes

Abstract

IncL/M broad-host-range conjugative plasmids are involved in the global spread of bla_OXA-48 and the emergence of bla_NDM-1. The aim of this study was to evaluate the transmission potential of plasmids encoding the emergent NDM-1 carbapenemase compared to the pandemic OXA-48. The conjugation rate and fitness cost of IncM2 and IncL plasmids encoding these carbapenemase genes were tested using a variety of host bacteria. Genomic analysis of uropathogenic Escherichia coli SAP1756 revealed that bla_NDM-1 was encoded on an IncM2 plasmid, which also harboured bla_TEM-1, ble_MBL and sul1 and was highly similar to plasmids isolated from the same geographical area. Conjugation experiments demonstrated that NDM-1 and OXA-48-carrying plasmids transfer successfully between different Enterobacterales species, both in vitro and in vivo. Interestingly, E. coli isolates tested as recipients belonging to phylogroups A, B1, D and F were able to receive IncM2 plasmid pSAP1756, while phylogroups B2, C, E and G were not permissive to its acquisition. In general, the IncL OXA-48-carrying plasmids tested transferred at higher rates than IncM2 harbouring NDM-1 and imposed a lower burden to their host, possibly due to the inactivation of the tir fertility inhibition gene and reflecting their worldwide dissemination. IncM2 plasmids carrying bla_NDM-1 are considered emergent threats that need continuous monitoring. In addition to sequencing efforts, phenotypic analysis of conjugation rates and fitness cost are effective methods for estimating the pandemic potential of antimicrobial resistance plasmids.

Keywords: Enterobacterales; IncL/M; NDM-1; OXA-48; antimicrobial resistance; carbapenemase; conjugation; plasmid.

Figure 1

Comparison of closely related IncL/M plasmids, aligned against the reference plasmid pNDM-HK, using BLAST Ring Image Generator (BRIG) [17]. Genbank accession numbers, bacterial hosts, plasmid sizes and ring characteristics: pNDM-HK (HQ451074, E. coli, 88,803 bp, innermost ring, reference), pNDM-OM (JX988621, Klebsiella pneumoniae, 87,185 bp, purple ring), pIT-Sma-01/2014 (MH722219, Serratia marcescens, 88,129 bp, green ring), pIT-Cfr-01/2015 (MH722216, Citrobacter freundii, 78,923 bp, yellow ring), pEsST2350_SE_NDM (CP031322, E. coli, 75,645 bp, blue ring) and pSAP1756 ((E. coli, 73,743 bp, red ring). The outermost ring shows the genes identified in the reference plasmid pNDM-HK and the diagram next to the innermost ring represents the GC content of the reference plasmid.

Figure 2

Conjugation frequency (CF) of IncL/M plasmids harbouring bla_NDM-1 or bla_OXA-48 genes, represented as the logarithm of transconjugants per donor obtained after 1 h conjugation at 37 °C on LB agar. (A) Conjugation from the original donors to E. coli MG1655 (rifampicin or nalidixic acid spontaneous mutants), or J53 in cases where donors were resistant to both rifampicin and nalidixic acid. Plasmids encoding NDM-1 but belonging to other incompatibility groups were added for comparison (pNDM-MAR and pNDM-KN, IncH and IncA/C, respectively). (B) Conjugation from the transconjugants from A in E. coli MG1655 (spontaneous mutant resistant to nalidixic acid) to E. coli MG1655 (spontaneous mutant resistant to rifampicin). Circles represent the result of individual experiments with NDM-1-carrying IncM2 plasmids (except control plasmid pCTX-M-3, which is NDM-1-negative) and squares represent CF with IncL plasmids encoding OXA-48. Horizontal lines show the mean and vertical lines the standard deviation of at least 4 independent experiments.

Figure 3

Conjugation frequency (CF) of IncM2 NDM-1-carrying plasmid pSAP1756 and IncL OXA-48-carrying plasmid pOXA48_1 using E. coli MG1655 resistant to nalidixic acid as donor and E. coli MG1655 resistant to rifampicin as recipient. Blue circles and orange squares represent the logarithm of transconjugants per donor obtained per experiment after 4 h conjugation in Galleria mellonella inoculated with ~10⁷ CFU of donors and recipients separately. Horizontal lines show the mean and vertical lines the standard deviation of at least 8 independent experiments.

Figure 4

Generation time of E. coli MG1655 (resistant to nalidixic acid) in hours, with (orange circles) or without (blue squares) IncL/M plasmids, calculated during the exponential phase of growth in M9 minimal media supplemented with glucose. The differential growth of the host without and with plasmid represents the burden of the plasmid under these conditions. Horizontal lines show the mean and vertical lines the standard deviation of at least 5 independent experiments. One-way analysis of variance with Dunnett’s post hoc test was performed between the bacterial hosts with plasmid versus the control without plasmid (*: p < 0.05; ns: no significant difference).