Prediction of Fluoroquinolone Susceptibility Directly from Whole-Genome Sequence Data by Using Liquid Chromatography-Tandem Mass Spectrometry To Identify Mutant Genotypes

Abstract

Fluoroquinolone resistance in Gram-negative bacteria is multifactorial, involving target site mutations, reductions in fluoroquinolone entry due to reduced porin production, increased fluoroquinolone efflux, enzymes that modify fluoroquinolones, and Qnr, a DNA mimic that protects the drug target from fluoroquinolone binding. Here we report a comprehensive analysis, using transformation and mutant selection, of the relative importance of each of these mechanisms for fluoroquinolone nonsusceptibility using as a model system. Our improved biological understanding was then used to generate 47 rules that can predict fluoroquinolone susceptibility in clinical isolates. Key to the success of this predictive process was the use of liquid chromatography-tandem mass spectrometry to measure the abundance of proteins in extracts of cultured bacteria, identifying which sequence variants seen in the whole-genome sequence data were functionally important in the context of fluoroquinolone susceptibility.

Resistance to Ceftazidime/Avibactam Plus Meropenem/Vaborbactam When Both are Used Together Achieved in Four Steps From Metallo-β-Lactamase NegativeKlebsiella pneumoniae

“…The highest MIC was against the ompK36 mutant, but it remained meropenem/vaborbactam susceptible. This was expected, since it has been shown that meropenem/vaborbactam resistance in derivatives of K. pneumoniae clinical isolates is primarily caused by ompK36 loss in a background where ompK35 has already been lost (19) and we have previously reported that Ecl8 actually produces more OmpK35 than clinical isolates (29).…”

“…K. pneumoniae clinical isolate KP21 produces low levels of OmpK35 (29). Experimentally, it has been shown that ramR loss of function downregulates OmpK35 production in K. pneumoniae (27) and KP21 carries a frameshift mutation in ramR (29) whilst whole genome sequencing confirmed that ompK35 itself is wild type.…”

“…K. pneumoniae clinical isolate KP21 produces low levels of OmpK35 (29). Experimentally, it has been shown that ramR loss of function downregulates OmpK35 production in K. pneumoniae (27) and KP21 carries a frameshift mutation in ramR (29) whilst whole genome sequencing confirmed that ompK35 itself is wild type. We introduced a natural OXA-232 plasmid into this isolate, and the MIC of meropenem in the presence of vaborbactam against KP21(pOXA-232) was 1 μg.mL −1 ( Table 2 ) which is below the resistance breakpoint and equivalent to our findings for Ecl8 ramR (pOXA-232) ( Table 1 ).…”

Mutation of kvrA causes OmpK35/36 porin downregulation and reduced meropenem/vaborbactam susceptibility in KPC-producing Klebsiella pneumoniae

“…Because FQ3 is derived from Ecl8, a laboratory strain, and because FQ3 carries mutations that increase efflux pump production (9), we wanted to test the impact of kvrA inactivation in a wild-type clinical isolate. To do this, we insertionally inactivated kvrA (as above) in the susceptible K. pneumoniae clinical isolate S17, which has wild-type envelope permeability (26) and showed by LC-MS/MS that OmpF and OmpC levels fell in this mutant relative to S17, as seen in FQ3. Porin abundance did not change upon nlpD inactivation in S17, also as seen in FQ3 (Fig.…”

“…In contrast, meropenem/vaborbactam MICs against the kvrA and ompK36 mutants were 1 µg.mL −1 (four doublings higher than against S17) and 32 µg.mL −1 respectively (Table 4). Hence, even in an otherwise wild-type KPC-producing clinical K. pneumoniae isolate (26), OmpK35/OmpK36 downregulation caused solely by kvrA mutation is enough to reduce meropenem/varborbactam susceptibility.…”