Comparative effects of overproducing the AraC-type transcriptional regulators MarA, SoxS, RarA and RamA on antimicrobial drug susceptibility inKlebsiella pneumoniae

Abstract

RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates.

Prediction of fluoroquinolone susceptibility directly from whole genome sequence data using liquid chromatography-tandem mass spectrometry to identify mutant genotypes

“…Cells in cultures were subjected to total envelope proteomics as described previously. 30 For total cell proteomics of K. pneumoniae bloodstream isolates, cells were cultured in nutrient broth (Oxoid) as above and lysed by sonication; a cycle of 1 sec on, 0.5 sec off for 3 min at amplitude of 63% using a Sonics Vibraceli VC-505TM (Sonics and Materials Inc., Newton, Connecticut, USA). Following centrifugation (15 min, 4°C, 8,000 rpm, Sorval RC5B with SS34 rotor), 1 μg of total protein was separated by SDS-PAGE.…”

“…The resulting peptides were fractionated using an Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). 30 The raw data files were processed and quantified using Proteome Discoverer software vl.4 (Thermo Scientific) and searched against the UniProt K. pneumoniae strain ATCC 700721 / MGH 78578 database (5126 protein entries; UniProt accession 272620) using the SEQUEST (Ver. 28 Rev.…”

The rapid spread of carbapenem-resistant Enterobacteriaceae

“…Overexpression of an additional AraC-type TR, ramA , in the K. pneumoniae NCTC 5055 reference strain increased growth around an imipenem Kirby-Bauer disk, suggesting a role in regulating carbapenem resistance [Jimenez-Castellanos 2016]. As reported in 2013, an E. coli isolate from China lacked OmpF and OmpC porins and had a marA mutation which resulted in expression of the previously classified pseudogene yedS [Warner 2013].…”

Prediction of cephalosporin and carbapenem susceptibility in multi-drug resistant Gram-negative bacteria using liquid chromatography-tandem mass spectrometry

“…The resulting peptides were fractionated using an Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) [18]. The raw data files were processed and quantified using Proteome Discoverer software v1.4 (ThermoScientific) and searched against the UniProt K. pneumoniae strain ATCC 700721 / MGH 78578 database (5126 protein entries; UniProt accession 272620), the P. aeruginosa PA01 database (5563 proteins; UniProt accession UP000002438), the A. baumannii ATCC 17978 database (3783 proteins; UniProt accession UP0006737) or the E. coli MG1655 database (4307 proteins; UniProt accession UP000000625), in each case, the strain-specific proteome database was augmented by addition of a mobile resistance determinant database (24694 proteins), which was generated by searching UniProt with “IncA”, “IncB” etc to “Inc-Z” as the search term and downloading each list of results.…”

“…This has recently been reviewed, and, whilst there have been some successes identifying some ABR proteins in some cases, a methodology to allow whole proteome analysis that can accurately quantify ABR proteins, which can be used to accurately predict antimicrobial susceptibility is yet to be demonstrated [17]. We have been using so called “shotgun” proteomics via a nano-liquid chromatography, Orbitrap tandem mass spectrometry approach to characterise proteomic responses to mutations in regulators that affect ABR [16,18]. Accordingly, without significant adaptation of the methodology, in the work reported here, we attempted to quantify and identify ABR proteins directly from clinical bacterial isolates grown in culture, and use this information to predict antimicrobial susceptibility across a range of species.…”