Vancomycin Heteroresistance Caused by Unstable Tandem Amplifications of the Gene Cluster on Linear Conjugative Plasmids in a Clinical

Vancomycin heteroresistance is prone to missed detection and poses a risk of clinical treatment failure. We encountered one clinical strain, SRR12, that carried a complete gene cluster but was determined as susceptible to vancomycin using the broth microdilution method. However, distinct subcolonies appeared within the clear zone of inhibition in the E-test assay, one of which, named SRR12-v1, showed high-level resistance to vancomycin. SRR12 was confirmed as heteroresistant to vancomycin using population analysis profiling and displayed "revive" growth curves with a lengthy lag phase of over 13 hours when exposed to 2-32 mg/L vancomycin. The resistant subcolony SRR12-v1 was found to carry an identical gene cluster to that of SRR12 but a significantly increased copy number in the genome. Long-read whole genome sequencing revealed that a one-copy gene cluster was located on a pELF1-like linear plasmid in SRR12. In comparison, tandem amplification of the gene cluster jointed with IS was seated on a linear plasmid in the genome of SRR12-v1. These amplifications of the gene cluster were demonstrated as unstable and would decrease accompanied by fitness reversion after serial passaging for 50 generations under increasing vancomycin pressure or without antibiotic pressure but were relatively stable under constant vancomycin pressure. Further, resistance in resistant variants was verified to be carried by conjugative plasmids with variable sizes using conjugation assays and S1-pulsed field gel electrophoresis blotting, suggesting the instability/flexibility of cluster amplification in the genome and an increased risk of resistance dissemination.