707. Clarifying the Role of CrrB in Polymxyin Resistant Klebsiella pneumoniae Clinical Isolates Utilizing a Novel CRISPR-Cas9 System
Session: Poster Abstract Session: Resistance Mechanisms: Gram-Negative
Thursday, October 4, 2018
Room: S Poster Hall

Background: Polymyxin resistance (PR) threatens the mainstay of therapy for carbapenem-resistant Enterobacteriaceae (CRE) infections. While mgrB disruption accounts for most cases of PR, missense mutations in crrB have been proposed as an alternative pathway for PR through PmrA/B/C upregulation of the pmrHFIJKLM operon. It remains unknown if CrrB acts as a positive or negative regulator on its downstream targets.

Methods: We assembled a CRISPR-Cas9 system for gene knockouts (KO) in CRE K. pneumoniae (CRKP) using zeocin as a selectable marker. We chose a polymyxin susceptible (PS) and a PR isolate with a missense mutation in crrB (L87V) (NR5337 and NR5083, respectively) for KO. Isolates were transformed with a crrB KO plasmid, grown with zeocin selection, induced with arabinose, and plated on low-salt LB-zeocin / arabinose. KOs were confirmed via PCR and Sanger sequencing. Polymyxin susceptibility was performed with broth-microdilution. Gene expression was determined by qRT-PCR of cDNA extracts.

Results: Colistin MIC following crrB KO of NR5337 (PS) remained unchanged. In contrast, crrB KO of NR5083 (PR), decreased polymyxin MIC (MIC >128 to 1.0 ug/ml). qRT-PCR of NR5083 did not show increased expression of pmrA/C, nor pmrK. NR5083 ^crrB showed a small decrease in phoQ expression, compared to NR5083, but similar expression of phoP, pmrA/C and pmrK (Table 1).

Conclusion: Polymyxin MIC decreased >128 fold after crrB KO in a PR isolate, but colistin MIC remained unchanged after KO in a PS isolate. CrrB mutations in PR isolates may confer a gain of function with CrrB acting as a positive regulator on its downstream targets. Contrary to previous literature, no upregulation of pmrA/C and pmrHFIJKLM was detected. Differences in crrB mutations or clonal background may explain this finding. CRISPR-Cas9 may serve as a reliable system for genetic manipulation of CRKP. Further data on the impact of individual crrB missense mutations are needed.

 Isolate Data

qRT-PCR Results

Strain

ST

CrrB Background

Colistin

MIC (E Test) ug/ml

Polymyxin B MIC (BMD) ug/ml

phoP

phoQ

pmrA

pmrC

pmrK

NR 5083

258

L87V

4.0

>128

3

2.3

0.8

0.8

0.9

NR 5083 ^crrb

258

160bp deletion

0.38

1

2.9

2.03

0.83

0.6

0.9

0.29

0.04

0.42

0.3

0.99

P* Value

NR 5337

258

WT

0.125

NR 5337 ^crrb

258

160bp deletion

0.125

Table 1: *T Test

Thomas McConville, MD, Marla Giddins, B.A., Nenad Macesic, MBBS and Anne-Catrin Uhlemann, MD, PhD, Columbia University Medical Center, New York, NY

Disclosures:

T. McConville, None

M. Giddins, None

N. Macesic, None

A. C. Uhlemann, Merck: Investigator , Grant recipient .

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