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

Abstract

In vitro antibacterial susceptibility testing informs clinical decision making concerning antibacterial therapeutics. Predicting, in a timely manner, which bacterial infection will respond to treatment by a given antibacterial drug reduces morbidity, mortality, and healthcare costs. It also allows prudent antibacterial use, because clinicians can focus on the least broad-spectrum agent suitable for each patient. Existing susceptibly testing methodologies rely on growth of bacteria in the presence of an antibacterial drug. There is significant interest in the possibility of predicting antibacterial drug susceptibility directly though the analysis of bacterial DNA or protein, because this may lead to more rapid susceptibility testing directly from clinical samples. Here we report a robust and tractable methodology that allows measurement of the abundance of key proteins responsible for antibacterial drug resistance within samples of 1 µg of total bacterial protein. The method allowed correct prediction of β-lactam susceptibility in clinical isolates from four key bacterial species and added considerable value over and above the information generated by whole genome sequencing, allowing for gene expression, not just gene presence to be considered, which is key when considering the complex interplays of multiple mechanisms of resistance.

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

“…We also report that inactivation of kvrA causes cefuroxime resistance in K. pneumoniae , but more importantly, it causes reduced susceptibility meropenem/vaborbactam, even in an otherwise wild-type K. pneumoniae clinical isolate, transformed to express bla KPC-3 from its native promoter in a low copy number vector (25). We show that cefuroxime resistance and reduced meropenem/vaborbactam susceptibility in a kvrA mutant is associated with OmpK35/OmpK36 downregulation.…”

“…To do this, kvrA DNA was amplified from K. pneumoniae Ecl8 genomic DNA by using primers (introduced restriction sites underlined): kvrA full length Bam HI FW (5’-AAA GGATCC CGGCAATCCGGATGTGTTAAGAC-3’) and kvrA full length SalI RV (5’-AAA GTCGAC GGAGGGTGAAAAAAGGCCCGGATTA-3’). The PCR product was digested and inserted to pUBYT (25) cut with BamHI and SalI to generate pUBYT:: kvrA . The recombinant plasmid was then transferred into FQ3 M1 cells by electroporation.…”

“…1C) on meropenem/vaborbactam susceptibility when the mutant produces KPC. To investigate this, we used pUBYT:: bla KPC-3 (25) to transform K. pneumoniae S17, S17Δ kvrA and S17Δ ompK36 (as a control). As expected, due to carriage of KPC, all transformants were resistant to meropenem.…”

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

“…bla CTX-M-14 was amplified from a human urinary E. coli isolated from primary care (38) by PCR with Phusion High-Fidelity DNA Polymerase (NEB, UK) using primers CTX-M-14 FW (5′-CCGGAATTCAATACTACCTTGCTTTCTGA-3′) and CTX-M-14 RV (5′-CCGGAATTCCGTAGCGGAACGTTCATCAG-3′) and ligated into pUBYT (39) at the EcoRI site. CTX-M-14 site-directed mutagenesis ( bla CTX-M-14-P170S ) was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, USA) with the primer CTX-M-14-P170S-FW (5′-TCTGGATCGCACTGAATCTACGCTGAATACCGC-3′).…”

Resistance to aztreonam in combination with non-β-lactam β-lactamase inhibitors due to the layering of mechanisms in Escherichia coli identified following mixed culture selection

“…7 The raw data files were processed and quantified using Proteome Discoverer software v1.4 (Thermo Scientific) and searched against bacterial genome and horizontally acquired resistance genes as described previously. 8…”