ESBL-Producing Escherichia coli and Klebsiella pneumoniae Exhibit Divergent Paths During In-Human Evolution Towards Carbapenem Resistance

Abstract

Treatment of infections caused by ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) with carbapenem antibiotics can lead to the development of carbapenem resistance over time through the acquisition of porin mutations and plasmids bearing blaKPC. However, the impact of genetic background and the presence of CRISPR-Cas systems on the evolutionary path towards carbapenem resistance in EC and KP has yet to be investigated. The in-human evolution following repeated carbapenem treatment among ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) clinical pairs (n = 45 pairs) was examined to determine the relationship between strain genetic background (MLST, CRISPR-Cas) and the evolved genetic mutations related to resistance, virulence, and metabolism by whole genome sequencing. ST131 and ST258 were predominant among seven distinct STs in EC (70%, 19/27) and 11 STs in KP (33%, 6/18), respectively. Complete CRISPR-Cas systems were present in 22% EC (6/27) and 27.8% (5/18) KP pairs, but none in strains belonging to ST131 or ST258; partial loss of CRISPR-Cas was associated with increased carbapenem resistance. Porin, virulence, and metabolism-related genetic mutations were present on the chromosome in both the EC and KP evolved strains, but their presence was differentially associated with the CRISPR-Cas system. Future research on the role of antibiotic exposure in the species-specific resistance evolution of the Enterobacterales could guide antimicrobial stewardship efforts.

Keywords: CRISPR-Cas; ESBL-producing Enterobacterales; carbapenem resistance; genetic evolution.

Figure 1

Resistance phenotypes of EC and KP pairs isolated from patients with repeated colonization or infection.

Figure 2

ESBL gene variants observed across index strains of (A) ESBL EC and (B) ESBL KP. Genomic reads from whole genome sequencing were assembled and analyzed using Comprehensive Genome Analysis by BV-BRC.

Figure 3

Multiple Locus Sequence Types (MLSTs) observed across evolved strains of (A) EC and (B) KP. Genomic reads from whole genome sequencing were assembled and annotated using Comprehensive Genome Analysis by BV-BRC. MLST assigned to strains as part of the analysis performed by the Comprehensive Genome Analysis program.

Figure 4

Protein sequence alignments of porins between index and evolved (A) EC and (B) KP pairs with identified mutations in the coding region of the genes. MAFFT alignments performed using Geneious software. Specific mutations identified in the evolved strain are noted in red text. Red boxes highlight single polymorphisms. (*) Denotes a stop codon in the sequence.

Figure 5

Summary of nonsynonymous mutations acquired in evolved EC and KP differentiated by the presence or absence of CRISPR-Cas system. Created in BioRender. Kalu, M. (2025). Images are representative of systems affected by nonsynonymous mutations found in evolved strains (see Table 2 and Table 3 for the specific genetic mutations identified). Cas −, absence of complete system; Cas +, presence of complete system.