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To understand the potential utility of novel nitroreductase (NR)-activated prodrugs, NR enzyme activity was assessed in clinical Klebsiella pneumoniae isolates using a NR-activated fluorescent probe. NR activity was constant throughout the bacterial growth cycle, but individual K. pneumoniae isolates exhibited a wide range of NR activity levels. The genes of major NR enzymes (nfsA and nfnB) showed a number of sequence variants. Aside from a C-terminal extension of NfnB, which may be responsible for lower NR activity in specific isolates, the genetic differences did not explain the variation in activity. Analysis of important clinical strains (ST11, ST258, ST14 and ST101) showed significant variation in NR activity between isolates within the same sequence type despite conservation of nfsA/nfnB sequences. Addition of methyl viologen (MV), a known activator of soxRS, caused a significant increase in NR activity for all strains, with proportionally larger increases in activity seen for strains with low uninduced NR levels. Real-time PCR on selected strains following exposure to MV showed upregulation of soxS (15-32-fold) and nfsA (5-22-fold) in all strains tested. Expression of nfnB was upregulated 2-5-fold in 4/6 strains tested. High levels of NR activity in the absence of MV activation correlated with nitrofurantoin susceptibility. These data provide evidence that NR gene mutations and regulatory pathways influence NR activity in K. pneumoniae isolates and this is likely to impact treatment efficacy with novel nitro-containing drugs or prodrugs., (Copyright © 2019. Published by Elsevier B.V.)

The lack of novel antibiotics for more than a decade has placed increased pressure on existing therapies to combat the emergence of multidrug-resistant (MDR) bacterial pathogens. This study evaluated the Galleria mellonella insect model in determining the efficacy of available antibiotics against planktonic and biofilm infections of MDR Pseudomonas aeruginosa and Klebsiella pneumoniae strains in comparison with in vitro minimum inhibitory concentration (MIC) determination. In general, in vitro analysis agreed with the G. mellonella studies, and susceptibility in Galleria identified different drug resistance mechanisms. However, the carbapenems tested appeared to perform better in vivo than in vitro, with meropenem and imipenem able to clear K. pneumoniae and P. aeruginosa infections with strains that had bla(NDM-1) and bla(VIM) carbapenemases. This study also established an implant model in G. mellonella to allow testing of antibiotic efficacy against biofilm-derived infections. A reduction in antibiotic efficacy of amikacin against K. pneumoniae and P. aeruginosa biofilms was observed compared with a planktonic infection. Ciprofloxacin was found to be less effective at clearing a P. aeruginosa biofilm infection compared with a planktonic infection, but no statistical difference was seen between K. pneumoniae biofilm and planktonic infections treated with this antibiotic (P>0.05). This study provides important information regarding the suitability of Galleria as a model for antibiotic efficacy testing both against planktonic and biofilm-derived MDR infections., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)