# Elucidating the genotypic determinants and phenotypic effects of polymyxin resistance in Klebsiella pneumoniae utilizing CRISPR-Cas9

> **NIH NIH K08** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2022 · $192,456

## Abstract

PROJECT SUMMARY / ABSTRACT: Rationale: Carbapenem resistant Enterobacteriaceae (CRE), especially
Klebsiella pneumoniae (KP) remain a significant public health threat. With a lack of treatment options, the
polymyxins remain a mainstay of therapy. The rise of polymyxin resistant KP (PRKP) threatens these vital
antibiotics. While modifications in the bacterial lipopolysaccharide (LPS) have proven the major mechanism of
PR, a wide range of mutations in three two components systems (TCS), phoP/Q, crrA/B, pmrA/B, and mgrB, are
thought to induce PR, but most have not been functionally validated. Additionally, little is known about how these
mutations affect bacterial fitness and virulence, and if they can increase polymyxin minimum inhibitory
concentration (MIC) independent of LPS modification. This mentored career development award aims to
elucidate the downstream phenotypic effects of a broad selection of mutations in these genes. Candidate: As
an infectious diseases clinician with a strong background in bacteriology and molecular biology, I am well suited
to pursue translational research focusing on the determinants of antibiotic resistance. Further training in bacterial
pathogenesis, anti-microbial resistance, bacterial genomics, and biostatistics will be crucial for the completion of
the proposed research and advancement of my career. With primary mentor Dr. Anne-Catrin Uhlemann, I have
assembled a multi-disciplinary team of experts to guide my training and research progress. My long-term goal is
to become an independent NIH-funded researcher utilizing novel molecular biology techniques to characterize
the determinants of bacterial antibiotic resistance and improve clinical practice. Environment: The Uhlemann
laboratory at Columbia University Irving Medical Center has the microbiology, molecular biology and sequencing
tools to complete the proposed research. The laboratory contains a large collection of CRE and PRKP clinical
isolates that have undergone whole genome sequencing. Columbia has a long track record of supporting the
career development of young investigators. Approach: Our central hypothesis is that the accumulation of
multiple mutations in the PR cascade leads to rising MICs and changes in bacterial virulence through activation
of unique cellular pathways. To elucidate the contribution of various mutations we will systematically insert these
into two CRKP clinical isolates utilizing our CRISPR-Cas9 system (Aim 1). We will characterize how these
changes affect MIC and LPS. In Aim 2 we will evaluate if PR can alter bacterial fitness and virulence through
growth curves, co-incubation analyses, Galleria mellonella killing assays and a mouse pneumonia model. In Aim
3 we will utilize RNA-seq to characterize the differential phenotypes of the PR mutants by defining the cellular
targets of the TCS. Through this we aim to identify novel pathways involved in PR and virulence and validate
these targets through CRISPR mediated modification. In addi...

## Key facts

- **NIH application ID:** 10431975
- **Project number:** 5K08AI146284-03
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Thomas Howe McConville
- **Activity code:** K08 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $192,456
- **Award type:** 5
- **Project period:** 2020-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10431975

## Citation

> US National Institutes of Health, RePORTER application 10431975, Elucidating the genotypic determinants and phenotypic effects of polymyxin resistance in Klebsiella pneumoniae utilizing CRISPR-Cas9 (5K08AI146284-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10431975. Licensed CC0.

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