Distribution of IncX3 plasmids in bacteria – Authors’ reply
Authors: Qiu E Yang, Shungui Zhou, Timothy R Walsh
Journal: The Lancet Microbe (2024)

The genomic analysis and argument presented by Yu Feng and Zhiyong Zong are interesting because they identified IncX3 in three bacteria belonging to the Gram-positive phylum Bacillota: Bacillus subtilis (GCF_000735115.1), Enterococcus faecalis (GCF_029906165.1), and Lactiplantibacillus plantarum (GCF_004102885.1). First, even if these genomes are incomplete, there is evidence to suggest that IncX3 is also present in species outside the Enterobacteriales order. We also analysed all available plasmid contigs in the NCBI nucleotide database (n=134 164; accessed on Jan 30, 2024), and 33 125 sequences were assigned to known Inc plasmids by PlasmidFinder (Galaxy Version 0.10.0+galaxy1). We identified IncX3 in species outside the Enterobacterales order, with seven of the bacteria belonging to Burkholderiales, namely six Ralstonia ornithinolytica and one Ralstonia planticola (appendix 1). However, we found that 99·66% of bacterial hosts of those plasmids were primarily assigned to two main phyla, Pseudomonadota (n=25 957, 78·36%) and Bacillota (n=7057, 21·30%), with the remaining hosts belonging to Bacteroidota (n=81, 0·25%) and Actinomycetota (n=9, 0·027%), which is contradictory to the diversity of bacterial phyla within an environmental habitat. Second, only genomes uploaded, by definition, come from culturable bacteria, and therefore, the dataset too is biased.1Pham VHT Kim J Cultivation of unculturable soil bacteria.Trends Biotechnol. 2012; 30: 475-484Summary Full Text Full Text PDF PubMed Scopus (0) Google Scholar Third, we showed that in enterococci, the plasmid is unstable2Yang QE Ma X Zeng L et al.Interphylum dissemination of NDM-5-positive plasmids in hospital wastewater from Fuzhou, China: a single-centre, culture-independent, plasmid transmission study.Lancet Microbe. 2024; 5: e13-e23Summary Full Text Full Text PDF Scopus (0) Google Scholar and that the number of hits derived from genomic mining, bias notwithstanding, are few. Regarding the concern by Feng and Zong on contamination, we present the following evidence. First, we repeated all non-culturable mating with Escherichia coli (pX3_NDM-5) and found identical results (appendix 2 p 1). The optimised gating strategy and agar plating results confirmed the high purity and accuracy of sorted transconjugants (appendix 2 p 1), and the broad transfer potential of the pX3_NDM-5 plasmid was reproducible (appendix 2 p 1). Second, we also repeated conjugation experiments between donor strain E coli MG1655 harbouring the plasmid pX3_NDM-5 and recipients from Bacillota and Bacteroidota (appendix 2 p 2). The gfp-expressing transconjugants were observed in all mating pairs, indicating that the pX3_NDM-5 plasmid has the potential to transfer across many different bacterial phyla. The limitations of culture-based conjugation are apparent; however, for many decades, the conjugation has dictated the host range of antimicrobial resistance (AMR) plasmids and has mitigated our understanding of how resistance spreads throughout natural bacterial communities.3Brito IL Examining horizontal gene transfer in microbial communities.Nat Rev Microbiol. 2021; 19: 442-453Crossref PubMed Scopus (106) Google Scholar,4Brockhurst MA Harrison E Ecological and evolutionary solutions to the plasmid paradox.Trends Microbiol. 2022; 30: 534-543Summary Full Text Full Text PDF PubMed Scopus (42) Google Scholar Our uniquely applied method challenges current and long-established paradigms on the spread of AMR plasmids and reveals a myriad of possibilities to truly examine the real-world distributions of AMR plasmids. We do not claim that our method is definitive; however, we are continually refining our method to completely understand the actual distribution of AMR across environmental, animal, and human One Health sectors. We declare no competing interests. This work was supported by the National Science Foundation of China (Grant No 32100150 and Grant No 42277436). Download .xlsx (2.93 MB) Help with xlsx files Supplementary appendix 1 Download .pdf (.36 MB) Help with pdf files Supplementary appendix 2