Phenotypic and multi-omics characterization ofEscherichia coliK-12 adapted to quaternary ammonium compounds identifies lipid A and cell envelope alterations regulated by mar-sox-rob and stress inducible pathways
Authors: Green Kari A.C. , Gregorchuk Branden S.J. , Reimer Shelby L. , Cartwright Nicola H. , Beniac Daniel R. , Hiebert Shannon L. , Booth Timothy F. , Slipski Carmine J. , Chong Patrick M. , Westmacott Garrett R. , Zhanel George G. , Bay Denice C. 
Published: July 2020

Quaternary ammonium compounds (QACs) benzalkonium (BZK) and cetrimide (CET) are common disinfectants used to inhibit or eradicate Gram-negative bacteria in clinical and agricultural products. QAC tolerance inEscherichia coliand other Enterobacterales species can confer cross-resistance to various clinically used antibiotics, making it important to understand mechanisms of QAC tolerance in greater depth. QAC adaptation byE. coliis hypothesized to alter MarRAB regulated genes that converge on the outer membrane, specifically, lipid A biosynthesis and transport genes, porins, and efflux pump systems. To test this, we performed a ‘multi’-omics and phenotypic characterization ofE. coliK-12 adapted to BZK and CET, to assess how QACs alter cell growth, genomics, and proteomics.E. coliadapted to either BZK and CET resulted in strains with stable QAC tolerance when either drug was omitted, elongated and narrower cell morphologies by scanning electron microscopy, and reduced growth fitness when compared to un-adaptedE. coli. Antimicrobial susceptibility testing revealed that QAC adaptation increasedE. colitolerance by ≥4-fold to BZK, CET, and other QACs but no antibiotic cross-resistance. Single nucleotide variants identified by whole genome sequencing and differentially accumulated proteins by liquid chromatography-mass spectrometry identified alterations to various QAC-adaptedE. coligenes and proteins belonging to: lipid A biosynthesis and transport (lpxLM, msbA, mla), themar-sox-robregulatory pathway (marR, rob), DNA/protein translation (gyrA, rpsA, rpoB, rapA). These alterations validate the hypothesis thatmar-sox-robnetwork plays a role in QAC tolerance and identifies additional stress inducible genetic and protein QAC tolerant biomarkers.

Importance

Bacterial tolerance mechanisms associated with disinfectant QAC adaptation is hypothesized to overlap with the mar-sox-rob multiple antimicrobial resistance pathway but has not been directly shown. Here, we generate QAC tolerantE. colistrains and identify phenotypic changes associated with protein and genetic alterations caused by prolonged QAC exposure. We identified genes that overlap with known antibiotic resistance mechanisms as well as distinct genes and proteins specific to QAC adaptation that are useful for future bacterial disinfectant tolerance mechanism studies. However, these altered genes and proteins implicate MarR and Rob pathways specifically in QAC tolerance but, surprisingly, the involvement of mar-sox-rob pathways did not increase antibiotic cross-resistance. Many altered genes we identified were essential genes in lipid A biosynthesis/transport, DNA and RNA transcription, and protein regulation systems potentially explaining why only QAC cross-tolerance was observed and why we observed greater cell fitness costs despite MarR and Rob pathway involvement.