Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

Abstract

Enterococci are naturally tolerant to typically bactericidal cell wall-active antibiotics, meaning that their growth is inhibited but they are not killed even when exposed to a high concentration of the drug. The molecular reasons for this extraordinary tolerance are still incompletely understood. Previous work showed that resistance to killing collapsed specifically in mutants affected in superoxide dismutase (Sod) activity, arguing that bactericidal antibiotic treatment led to induction of a superoxide burst. In the present work, we show that loss of antibiotic tolerance in ⌬sodA mutants of pathogenic enterococci is dependent on the energy source present during antibiotic treatment. Hexoses induce greater killing than the pentose ribose, and no killing was observed with glycerol as the energy source. These results point to glycolytic reactions as crucial for antibiotic-mediated killing of ⌬sodA mutants. A transposon mutant library was constructed in Enterococcus faecalis ⌬sodA mutants and screened for restored tolerance of vancomycin. Partially restored tolerance was observed in mutants with transposon integrations into intergenic regions upstream of regulators implicated in arginine catabolism. In these mutants, the arginine deiminase operon was highly upregulated. A model for the action of cell wall-active antibiotics in tolerant and nontolerant bacteria is proposed. IMPORTANCEAntibiotic tolerance is a serious clinical concern, since tolerant bacteria have considerably increased abilities to resist killing by bactericidal drugs. Using enterococci as models for highly antibiotic-tolerant pathogens, we showed that tolerance of these bacteria is linked to their superoxide dismutase (Sod), arguing that bactericidal antibiotics induce generation of reactive oxygen species inside cells. Wild-type strains are tolerant because they detoxify these deleterious molecules by the activity of Sod, whereas Sod-deficient strains are killed. This study showed that killing depends on the energy source present during treatment and that an increase in arginine catabolism partially restored tolerance of the Sod mutants. These results are used to propose a mode-of-action model of cell wall-active antibiotics in tolerant and nontolerant bacteria. Even though enterococci are considered low-virulence pathogens, treatment of enterococcal infections is often difficult and lengthy. International guidelines for treatment of infective enterococcal endocarditis recommend 4 to 6 weeks of administration of penicillin or ampicillin plus an aminoglycoside, with a risk of acute renal failure due to the nephrotoxicity of the latter antibiotics (1-3). Enterococci, which are lactic acid bacteria and part of the human commensal flora, are naturally highly tolerant to antibiotics considered to be bactericidal such as penicillins and glycopeptides (4, 5). The mechanisms that allow these organisms to escape the lethal action of those drugs are still incompletely understood. On the basis of previous studies, it was suggested that the tolerance...

Sublethal Paraquat Confers Multidrug Tolerance in Pseudomonas aeruginosa by Inducing Superoxide Dismutase Activity and Lowering Envelope Permeability

“…These results thus further support the important contribution of SOD activity to antibiotic tolerance, which is growth phase specific and negligible under rapid growth conditions as the absence of SOD activity does not affect antibiotic tolerance in exponentially growing P. aeruginosa. Our results with the sodAB mutant are consistent with studies in Enterococcus faecalis (Bizzini et al, 2009;Ladjouzi et al, 2015), Campylobacter jejuni (Hwang et al, 2013), Acinetobacter baumannii (Heindorf et al, 2014), and E. coli (Dwyer et al, 2007;Wang et al, 2014), where loss of SODs also enhances bactericidal antibiotic killing. These stand in contrast to other studies performed in exponentially growing E. coli reporting that the sodA sodB mutant exhibited similar susceptibility to ampicillin, gentamicin, and norfloxacin killing to the wild-type strain (Wang and Zhao, 2009;Ezraty et al, 2013), and that overexpression of sodA or sodB did not mitigate ampicillin and ofloxacin killing (Orman and Brynildsen, 2016).…”

“…While several groups have previously reported that bactericidal antibiotics induce production of reactive oxygen species, including superoxide and hydroxyl radicals, which contribute to their off-target killing mechanism ( Dwyer et al, 2007 ; Grant et al, 2012 ; Sampson et al, 2012 ; Imlay, 2013 ; Van Acker et al, 2016 ), others have refuted these observations ( Keren et al, 2013 ; Liu and Imlay, 2013 ). Superoxide stress also induces anti-oxidant defenses such as superoxide dismutases (SOD), which in turn modulate antibiotic lethality as we and others have reported ( Bizzini et al, 2009 ; Hwang et al, 2013 ; Ladjouzi et al, 2013 , 2015 ; Heindorf et al, 2014 ; Wang et al, 2014 ; Martins et al, 2018 ). We recently demonstrated that induction of SOD activity confers multidrug tolerance to stationary phase P. aeruginosa through alteration of the cell envelope permeability and increased drug accumulation ( Martins et al, 2018 ).…”

Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo

“…ATP can be generated when carbamoyl phosphate is dephosphorylated by carbamate kinase (31,32). This pathway is known to be particularly active in response to environmental stress (33,34) and is of special interest since poly(P) accumulation is associated with bacterial stress resistance. The levels of arginine, citrulline, and ornithine were higher in the ppk2::Tn and ppx1::Tn mutants than in the wild type (Fig.…”

Ctt1 catalase activity potentiates antifungal azoles in the emerging opportunistic pathogen Saccharomyces cerevisiae

“…This can be reversed by overexpression in the mutant of KatA, the dominant P. aeruginosa catalase 4 , or by restoration of Sod activity 22 . Many other bacteria become more susceptible to antibiotics on Sod deletion, including Enterococcus faecalis 9,23,24 , Campylobacter jejuni 8 , Acinetobacter baumanii 7 , Staphylococcus aureus 24 and E. coli in stationary-phase 25 but maybe not in exponentially growing cultures 15 . Deletion of the catalase-peroxidase katG or the alkyl hydroperoxide reductase ahpC also potentiates some antibiotics in E. coli 15 .…”