Distribution of IncX3 plasmids in bacteria
Authors: Yu Feng, Zhiyong Zong
Journal: The Lancet Microbe (2024)

A recent publication concluded that the IncX3 plasmid has broad host range.1Yang 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. 2023; 5: e13-e23Summary Full Text Full Text PDF PubMed Scopus (1) Google Scholar Although the finding is intriguing, it needs to be confirmed by studies using other methodologies or analyses from different directions. In particular, we are aware that the finding is inconsistent with the historical understanding that dates back to the early 1970s when the IncX plasmid R6K, characterised as narrow spectrum, was discovered.2Olsen RH Wright CD Interaction of Pseudomonas and Enterobacteriaceae plasmids in Aeromonas salmonicida.J Bacteriol. 1976; 128: 228-234Crossref PubMed Scopus (7) Google Scholar The IncX3 plasmid was first reported in 2012,3García-Fernández A Villa L Carta C et al.Klebsiella pneumoniae ST258 producing KPC-3 identified in Italy carries novel plasmids and OmpK36/OmpK35 porin variants.Antimicrob Agents Chemother. 2012; 56: 2143-2145Crossref PubMed Scopus (0) Google Scholar,4Johnson TJ Bielak EM Fortini D et al.Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae.Plasmid. 2012; 68: 43-50Crossref PubMed Scopus (218) Google Scholar and the first blaNDM−1-carrying IncX3 plasmid was also described in 2012 in China.5Ho PL Li Z Lo WU et al.Identification and characterization of a novel incompatibility group X3 plasmid carrying blaNDM−1 in Enterobacteriaceae isolates with epidemiological links to multiple geographical areas in China.Emerg Microbes Infect. 2012; 1: e39Crossref PubMed Scopus (0) Google Scholar Despite subsequent studies expanding on the known resistance and host range of IncX3 plasmids, none of the studies reported their existence in species beyond the Enterobacterales order. The large number of bacterial genome deposits in the NCBI is an important source, and we mined genomes from the NCBI to understand the distribution of IncX3 plasmids. We analysed all available bacterial assemblies in the NCBI RefSeq collection (n=325 562; accessed on Dec 9, 2023). Using the BLASTN algorithm against the 374-bp IncX3 replicon-typing sequences from PlasmidFinder, we defined the presence of IncX3 on the basis of a minimum nucleotide identity of 90% and coverage of 10%. Additionally, we cross-referenced the taxonomy assignments of these genomes with the GTDB database (release 08-RS214), which led to the exclusion of 557 assemblies that were deemed low quality according to the criteria outlined on the GTDB website, and found 325 005 assemblies for the downstream analysis. Our analysis identified 4510 IncX3-positive assemblies, of which only five were not part of the Enterobacterales order (appendix 1): Acinetobacter seifertii (GCF_001990725.1), Bacillus subtilis (GCF_000735115.1), Enterococcus faecalis (GCF_029906165.1), Lactiplantibacillus plantarum (GCF_004102885.1), and Legionella pneumophila (GCF_003205035.1). To verify the presence of the IncX3 plasmid within these assemblies, we conducted a second BLASTN search against the full-length 1014-bp pir gene sequence encoding the replication initiation protein of the IncX3 replicon (accession number JN247852.1). A seifertii, L plantarum, and L pneumophila strains showed only partial coverage of the full-length pir sequence, and 70·12% of A seifertii, 25·35% of L plantarum, and 10·16% of L pneumophila did not support the presence of the complete replicon (appendix 2 p 1). The remaining two strains contained the complete pir gene, but the existence of an IncX3 plasmid was not confirmed. First, only draft genome assemblies are available, and whether the replicon is incorporated into the chromosome or other plasmids remains unknown. Second, both B subtilis and E faecalis are commonly studied and sequenced, as evidenced by 693 RefSeq assemblies for B subtilis and 3087 for E faecalis. Apart from a recent study mentioning an IncX3 plasmid in E faecalis,1Yang 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. 2023; 5: e13-e23Summary Full Text Full Text PDF PubMed Scopus (1) Google Scholar no other cases have been documented. Third, the E faecalis strain also contains an IncFIB(K) replicon, generally considered to be an Enterobacteriaceae-specific one. Fourth, the genome of the E faecalis strain has a contamination score of 6·32 in the CheckM analysis, suggesting potential contamination of other sequences. Therefore, we believe that for both B subtilis and E faecalis, re-sequencing and obtaining complete genome assembly are crucial to eliminate the possibilities of contamination or the integration of the IncX3 replicon into other genomic regions. If subsequent studies confirm the presence of an IncX3 plasmid in non-Enterobacterales species, the findings will advance the understanding towards the host range of IncX3 plasmids and provide evidence to suggest their interphylum transmission. We declare no competing interests. The work was supported by a joint grant from the National Natural Science Foundation of China (grant no. 82002177 and 81861138055) and a grant from West China Hospital of Sichuan University (ZYYC08006). Download .xlsx (1.19 MB) Help with xlsx files Supplementary appendix 1 Download .pdf (.11 MB) Help with pdf files Supplementary appendix 2