Adaptive laboratory evolution and proteomic insight on collateral sensitivity for meropenem in colistin-resistant...

Antimicrobial resistance (AMR) is a critical global challenge, with an estimated ≥10 million deaths per year by 2050. Klebsiella pneumoniae (K. pneumoniae), a critical priority bacterial pathogen, is rapidly developing resistance even against next-generation (meropenem) and last-resort (colistin) antibiotics, whereas the pipeline for discovery and development of new antibiotic(s) for treatment of antibiotic-resistant K. pneumoniae infections is a lengthy, resource-intensive, and costly process and hence demands exploring alternative and effective treatment strategies. Capitalizing on the simultaneous emergence of collateral sensitivity (CS) to resistant antibiotics can therefore be a rapid and effective strategy to fill the gap.

Adaptive laboratory evolution was enforced in K. pneumoniae (American Type Culture Collection [ATCC] and clinical strains) by exposing it to sequentially increasing sub-lethal dosages (0.25, 0.5, and 1xMIC) of colistin and meropenem, and a panel of pathogens representing early (0.25xMIC), mid (0.5xMIC), and late (1xMIC) stages of emergence of AMR was developed. Similarly, colistin (1xMIC)-resistant K. pneumoniae cells were also adapted to develop resistance against meropenem. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) for pathogen identification and VITEK-2 for antibiotic susceptibility testing (AST) profiles validated resistant pathogens, which were then proteomic profiled to investigate CS between colistin and meropenem at both phenotypic and proteome levels.

AST of developed panels revealed that colistin-resistant pathogens became sensitive to meropenem during adaptation to colistin, representing CS, which was validated by the emergence of colistin sensitivity when colistin-resistant K. pneumoniae adapted against meropenem. Proteomic analysis of the resistant panels identified a total of 3,126 proteins, including 23 and 5 differentially expressed proteins (DEPs) in colistin- and meropenem-resistant pathogens, respectively. Among these, 12 proteins (phoP, arnB/arnD/arnT, and pmrD/pmrA) (associated with two components: system regulator and antibiotic resistance); degP, kdpB, maeA, frmR, fhuC, and entF (responsible for virulence and stress adaptation); and fhuC, entF, and amn (required for iron acquisition and persistence) showed altered pattern matching along with CS, that is, upregulated during emergence of colistin resistance along with meropenem sensitivity and conversely downregulated during emergence of colistin sensitivity along with meropenem-resistance in colistin-1x resistant K. pneumoniae.

This study, for the first time, developed a unique panel of colistin- and meropenem-resistant K. pneumoniae cells representing the early, mid, and late stages of AMR emergence. The simultaneous emergence of CS for meropenem while developing resistance against colistin among the panels may pave the way to devise antibiotic cycling and/or co-dosing approaches, and modulation of the expression of the identified 12 potential proteomic biomarkers may be utilized to regulate the emergence of CS for meropenem in colistin-resistant pathogens for combating colistin-resistant infections.