Genomic characterisation of multi-drug resistant Escherichia coli and Klebsiella pneumoniae co-harbouring mcr-1 and mcr-3 genes on a single plasmid from paediatric clinical cases.

Objectives: Emergence of the plasmid-born mobile colistin resistance (mcr) gene is a growing concern in healthcare. Therefore, this study aimed to genomically characterise multidrug-resistant Escherichia coli and Klebsiella pneumoniae co-harbouring the mcr-1 and mcr-3 genes in young children.

Methods: E. coli (n = 3) and K. pneumoniae (n = 2) were collected from abdominal secretions and blood, respectively. The isolates were screened using tryptone soy broth with 4 µL/mL polymyxin-B. Growing bacteria were identified using the VITEK-2 system, matrix-assisted laser desorption/ionisation time-of-flight, and 16s RNA sequencing, followed by antibiotic susceptibility testing. Metallo-β-lactamase (MBL) and extended-spectrum β-lactamase (ESBL) production was also detected. Afterwards, strains were subjected to molecular screening targeting mcr variants and ESBL/MBL-encoding genes. Conjugation, pulsed-field gel electrophoresis, Southern hybridisation, multilocus sequence typing, and phylogenic group detection were performed, along with plasmid-genome sequencing and bioinformatics analysis.

Results: E. coli isolates (EC-19-322, 323, and 331) and K. pneumoniae isolates (KP-19-225 and 226) harboured both mcr-1 and mcr-3 genes. These strains were also found to be resistant to more than three classes of antibiotics. The conjugation experiment revealed the presence of mcr-1 and mcr-3 on a single plasmid, and the transmission frequency was 10 to 10. Both strains were found to be able to produce ESBLs and MBL. E. coli EC-19-322 and 323 were identified as ST131(O25a:H41); SP-19-331, as ST1577 (O16:H30); and K. pneumoniae, as ST231 (K2). All E. coli strains belonged to phylogenetic group B2, and the results of pulsed-field gel electrophoresis supported the multilocus sequence typing findings.

Conclusion: This study reported the co-occurrence of mcr-1 and mcr-3 genes on a single plasmid in pathogenic ESBL/MBL-producing E. coli and K. pneumoniae isolated from young children.