# Phenotypic and genomic characterization of tigecycline heteroresistance in carbapenem-resistant Klebsiella pneumoniae

## TL;DR

This study explores tigecycline heteroresistance in drug-resistant Klebsiella pneumoniae, showing it's stable and linked to multiple resistance and virulence genes, which could complicate treatment.

## Contribution

The study systematically characterizes tigecycline heteroresistance in CRKP isolates, revealing its genomic basis and stable resistance without antibiotic exposure.

## Key findings

- Heteroresistant subclones of CRKP can survive at high tigecycline concentrations and maintain resistance without antibiotic exposure.
- Genomic analysis reveals co-occurrence of resistance genes like tet(A) and virulence factors, suggesting enhanced pathogenicity.
- Multireplicon plasmids in isolates may facilitate the spread of resistance genes, indicating a potential public health risk.

## Abstract

The presence of tigecycline heteroresistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) can compromise the efficacy of antimicrobial therapy. This study aimed to investigate the phenotypic and genomic characteristics of Tgc-HR in CRKP clinical isolates. A total of 52 non-repetitive CRKP isolates, all tigecycline-susceptible, were collected from January 2021 to December 2023. Tigecycline heteroresistance was confirmed by K-B disk diffusion, E-test screening, growth curves, population analysis profiling (colony pattern analysis), and time-kill curve in this study. Protein fingerprinting clustering, WGS analysis, and bioinformatics were used to investigate the phenotypic characteristics and resistance genes, virulence factors, and plasmid profiles of the isolates. The heteroresistant subclones could proliferate at tigecycline levels as high as 16 mg/L, and their resistant phenotype stayed stable even after repeated passages without antibiotic exposure. Analysis of protein fingerprint clustering suggested that the heteroresistant subclones were derived directly from the original strain, not from contamination by an external strain. All isolates were identified as ST-11 through multilocus sequence typing. The genomic analysis showed that the isolates contained numerous resistance genes, especially the tetracycline efflux pump gene tet(A), β-lactamase genes (blaTEM and blaCTX-M), the aminoglycoside-modifying enzyme gene (aadA), and the efflux pump regulators ramR and acrR. These resistance genes were found to co-occur with virulence factors, such as the type 3 fimbriae (mrk-associated loci), based on genomic co-occurrence analysis (which involves assessing the presence of genes in close proximity or co-localization on the genome), suggesting that resistance and virulence together could heighten pathogenicity and make treatment outcomes more challenging in clinical scenarios. The plasmid typing results indicated that the isolates contained a multireplicon plasmid capable of carrying several resistance genes and closely resembling known high-risk resistant plasmids, aiding in the dissemination of resistance. In this study, we systematically investigated the phenotypic and genomic features of tigecycline heteroresistance among clinical CRKP isolates in China and demonstrated its stability and plausibly genetic basis and hinted at treatment implications. The findings are predictions based off of genomic data estimations and have not been confirmed by transcriptomic nor functional research. More work is required to validate these mechanisms.

This study provides crucial insights into the phenomenon of tigecycline heteroresistance (Tgc-HR) in carbapenem-resistant Klebsiella pneumoniae. It highlights the stability of heteroresistant subpopulations and their ability to persist without selective antibiotic pressure, complicating treatment outcomes. The research shows that Tgc-HR is linked to multiple resistance and virulence factors, suggesting a co-evolution of resistance and pathogenicity. This finding underscores the importance of monitoring for heteroresistance in clinical settings, as it may lead to undetected therapeutic failures. Additionally, the study contributes to understanding the role of plasmids in the spread of resistance, emphasizing the need for comprehensive genomic surveillance to inform better antimicrobial strategies.