Mobile colistin resistance mcr-4.3- and mcr-4.6-harbouring plasmids in livestock- and human-retrieved Enterobacterales in the Netherlands

the mobile colistin resistance gene mcr-1 in 2015, 10 mcr variants were identified among antibiotic-resistant Enterobacterales and were mostly located on

After the first report of the mobile colistin resistance gene mcr-1 in 2015, 10 mcr variants were identified among antibiotic-resistant Enterobacterales and were mostly located on plasmids. 1,2 The mcr genes encode phosphoethanolamine transferases capable of modifying Lipid A in membrane-associated LPS, thereby ultimately leading to colistin resistance. 3 Colistin is considered a last-resort antibiotic for treatment of human Enterobacterales infections, but was also being used in veterinary medicine in the Netherlands in 2021. 4 Until now, the presence of mcr-4 has not been described before in The Netherlands. The major objective of this study was to analyse mcr-4-encoding plasmids from Enterobacterales obtained from humans and livestock in the Netherlands. To address this, a search for mcr-4-containing Enterobacterales isolates was performed in two surveillance databases containing Illumina short-read next-generation sequencing (NGS) data. These include the carbapenemase-producing Enterobacterales (CPE) surveillance collection from the National Institute for Public Health and the Environment (RIVM) and the livestock resistance monitoring collection from Wageningen Bioveterinary Research (WBVR). In addition, two clinical isolates of the Zuyderland Medical Center (ZMC) were analysed by the Maastricht University Medical Centre (MUMC).
To test mcr-4-mediated colistin resistance, two strategies were employed.
Firstly, cloning of mcr-4.3 and mcr-4.6 with and without native promotor using primers MCR4_ownpromotorF: CACGGGCAAAG ATCGGAGGG, MCR4_reverse: TCAGATCTCGTTGTAATTTTCAAGG and MCR4_withoutownpromotorF: GAGGTCAAGCTTGTATTGTTTTT into pGEM ® -T Easy (Invitrogen) in E. coli DH10β was performed according to the manufacturer's instructions. Plating was done on LB agar plates with 0, 0.125, 0.25, 0.5, 1 or 2 mg/L colistin and with ampicillin (50 mg/L). To avoid overexpression of the mcr-4 genes, IPTG was omitted in the LB agar plates (no blue-white screening). No colonies were found after overnight incubation at 37°C on plates containing colistin. Also, induction with IPTG for the native and non-native mcr-4 promotor constructs, did not result in colistin resistant colonies. Seven of the eight selected colonies that grew on LB agar plates with only ampicillin showed inverted mcr-4 inserts, as determined by sequencing.
Secondly, transformation of an Acinetobacter baumannii (BioSample: ERS7182715, MIC colistin 0.5 mg/L) with mcr-4.3 and mcr-4.6 plasmid DNA isolated from isolate MUMC-1 and WUR_NRS20181383, respectively, was performed. After transformation of A. baumannii with either mcr-4.3 or mcr-4.6 plasmids, only colonies were observed on plates with colistin concentration up to and including 0.25 mg/L, indicating there was no change in colistin resistance for this strain.
In summary, the occurrence of mcr-4 plasmids among Enterobacterales is low in the Netherlands. The mcr-4.3 allele in E. kobei and the mcr-4.6 allele in livestock E. coli and H. paralvei likely do not encode for colistin resistance in the human and livestock isolates. A recent study also failed to detect colistin resistance in E. kobei ST54 co-harbouring mcr-4.3 and mcr-9 and is in line with this study. 15 In contrast, mcr-4.3 conferred colistin resistance in A. baumannii and Acinetobacter nosocomialis, 16,17 suggesting species-specific functionality of this colistin resistance gene, but could not be confirmed in the A. baumannii isolate analysed in this study. The mcr-4 plasmids from human and livestock were virtually identical, suggesting unnoticed horizontal dissemination of these plasmids in the Netherlands.