Characterisation of mcr-4.3 in a colistin-resistant Acinetobacter nosocomialis clinical isolate from Cape Town, South Africa

Objectives

Colistin resistance in Acinetobacter spp. is increasing, causing potentially untreatable nosocomial infections. Plasmid-mediated colistin resistance is of particular concern due to its low fitness cost and potential transferability to other strains and bacterial species. This study investigated the colistin resistance mechanism in a clinical Acinetobacter nosocomialis isolate from Cape Town, South Africa.

Methods

A colistin-resistant A. nosocomialis isolate was identified from a blood culture in 2017. PCR and whole-genome sequencing (WGS), using Illumina, were performed to identify genes and mutations conferring colistin resistance. Plasmid sequencing was performed on the Oxford Nanopore platform. mcr functionality was assessed by BMD after cloning the mcr gene into pET-48b(+), expressing it in SHuffle T7 E. coli, and curing the plasmid using 62.5 mg/L acridine orange.

Results

The colistin MIC of the A. nosocomialis isolate was 16 mg/L. mcr-4.3 was detected by PCR and WGS. No other previously described colistin resistance mechanism was found by WGS. mcr-4.3 was identified on a 24 024 bp RepB plasmid (pCAC13a). Functionality studies showed that recombinant mcr-4.3 does not confer colistin resistance in E. coli. However, plasmid curing of pCAC13a restored colistin susceptibility in A. nosocomialis.

Conclusions

We describe the first detection of a plasmid-mediated mcr-4.3 gene that encodes colistin resistance in A. nosocomialis and the first detection of mcr-4.3 in a clinical isolate in Africa. Recombinant expression of mcr-4.3 did not confer colistin resistance in E. coli, suggesting that its functionality may be RepB plasmid dependent or species specific.