Clinical and Genomic Epidemiology of mcr-9-Carrying Carbapenem-Resistant Enterobacterales Isolates in Metropolitan Atlanta, 2012 to 2017

Abstract

Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative infections. Recently, the ninth allele of the mobile colistin resistance (mcr) gene family, designated mcr-9, was reported. However, its clinical and public health significance remains unclear. We queried genomes of carbapenem-resistant Enterobacterales (CRE) for mcr-9 from a convenience sample of clinical isolates collected between 2012 and 2017 through the Georgia Emerging Infections Program, a population- and laboratory-based surveillance program. Isolates underwent phenotypic characterization and whole-genome sequencing. Phenotypic characteristics, genomic features, and clinical outcomes of mcr-9-positive and -negative CRE cases were then compared. Among 235 sequenced CRE genomes, 13 (6%) were found to harbor mcr-9, all of which were Enterobacter cloacae complex. The median MIC and rates of heteroresistance and inducible resistance to colistin were similar between mcr-9-positive and -negative isolates. However, rates of resistance were higher among mcr-9-positive isolates across most antibiotic classes. All cases had significant health care exposures. The 90-day mortality was similarly high in both mcr-9-positive (31%) and -negative (7%) CRE cases. Nucleotide identity and phylogenetic analysis did not reveal geotemporal clustering. mcr-9-positive isolates had a significantly higher number of median [range] antimicrobial resistance (AMR) genes (16 [4 to 22] versus 6 [2 to 15]; P < 0.001) than did mcr-9-negative isolates. Pangenome tests confirmed a significant association of mcr-9 detection with mobile genetic element and heavy metal resistance genes. Overall, the presence of mcr-9 was not associated with significant changes in colistin resistance or clinical outcomes, but continued genomic surveillance to monitor for emergence of AMR genes is warranted. IMPORTANCE Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative infections. A recently described allele of the mobile colistin resistance (mcr) gene family, designated mcr-9, has been widely reported among Enterobacterales species. However, its clinical and public health significance remains unclear. We compared characteristics and outcomes of mcr-9-positive and -negative CRE cases. All cases were acquired in the health care setting and associated with a high rate of mortality. The presence of mcr-9 was not associated with significant changes in colistin resistance, heteroresistance, or inducible resistance but was associated with resistance to other antimicrobials and antimicrobial resistance (AMR), virulence, and heavy metal resistance (HMR) genes. Overall, the presence of mcr-9 was not associated with significant phenotypic changes or clinical outcomes. However, given the increase in AMR and HMR gene content and potential clinical impact, continued genomic surveillance of multidrug-resistant organisms to monitor for emergence of AMR genes is warranted.

Keywords: AMR; CRE; MDR; antimicrobial resistance; carbapenem-resistant Enterobacterales; healthcare epidemiology; multidrug resistance; next-generation sequencing.

FIG 1

Carbapenem-resistant Enterobacterales (CRE) count (A) and crude annual incidence per 100,000 population (B) by species across the Georgia Emerging Infections Program from 2012 to 2017. Beginning in 2016, the phenotypic CRE case definition was changed to resistance to ≥1 carbapenem (now including ertapenem) with no cephalosporin parameter.

FIG 2

Phylogeny (left) and average nucleotide identity heatmap (right) of mcr-9-positive (n = 13) and mcr-9-negative (n = 14) E. cloacae complex genomes from the Georgia Emerging Infections Program in addition to 9 available E. cloacae complex genomes (three mcr-9 positive, six mcr-9 negative) from the National Institutes of Health. A phylogenetic tree based on a core gene alignment containing 1,904 genes defined using Roary v3.13.0 was generated using IQtree v2.0.3. A maximum likelihood tree was generated by running 1,000 bootstrap replicates under the generalized time-reversible model of evolution. The tree was visualized and annotated using Interactive Tree of Life (iTOL) v4. Pairwise comparisons of average nucleotide identity on the assembled genomes were performed with the Mashmap method using fastANI v1.32. GA EIP, Georgia Emerging Infections Program; NIH, National Institutes of Health.

FIG 3

Antimicrobial resistance gene heatmap of mcr-9-positive (n = 13) and mcr-9-negative (n = 14) E. cloacae complex genomes from the Georgia Emerging Infections Program in addition to 9 available E. cloacae complex genomes (three mcr-9 positive, six mcr-9 negative) from the National Institutes of Health. Genomes were annotated using Prodigal v2.6.3, and antimicrobial resistance gene content was assessed using AMRFinder. Antimicrobial resistance gene presence/absence heatmaps were created using the package pheatmap on R version 4.0.2 (Vienna, Austria) and the RStudio interface version 1.3.1073 (Boston, MA, USA).