Antibiotic killing of diversely generated populations of non-replicating bacteria

Abstract

Non-replicating bacteria are known to be (or at least commonly thought to be) refractory to antibiotics to which they are genetically susceptible. Here, we explore the sensitivity to killing by bactericidal antibiotics of three classes of non-replicating populations of planktonic bacteria; (1) stationary phase, when the concentration of resources and/or nutrients are too low to allow for population growth; (2) persisters, minority subpopulations of susceptible bacteria surviving exposure to bactericidal antibiotics; (3) antibiotic-static cells, bacteria exposed to antibiotics that prevent their replication but kill them slowly if at all, the so-called bacteriostatic drugs. Using experimental populations of Staphylococcus aureus Newman and Escherichia coli K12 (MG1655) and respectively 9 and 7 different bactericidal antibiotics, we estimate the rates at which these drugs kill these different types of non-replicating bacteria. Contrary to the common belief that bacteria that are non-replicating are refractory to antibiotic-mediated killing, all three types of non-replicating populations of these Gram-positive and Gram-negative bacteria are consistently killed by aminoglycosides and the peptide antibiotics, daptomycin and colistin, respectively. This result indicates that non-replicating cells, irrespectively of why they do not replicate, have an almost identical response to bactericidal antibiotics. We discuss the implications of these results to our understanding of the mechanisms of action of antibiotics and the possibility of adding a short-course of aminoglycosides or peptide antibiotics to conventional therapy of bacterial infections.

Eradicating Bacterial Persisters with Combinations of Strongly and Weakly Metabolism-Dependent Antibiotics

“…In parallel, we tested colistin and mitomycin C as molecules reported to demonstrate bactericidal efficacy against metabolically repressed cells (McCall et al, 2019;Grassi et al, 2017;Kwan et al, 2015). Consistent with previous literature (Lopatkin et al, 2019;Lobritz et al, 2015;Gutierrez et al, 2017;McCall et al, 2019;Grassi et al, 2017;Kwan et al, 2015), ampicillin and ciprofloxacin lost significant bactericidal efficacy against nutrientdepleted cells, whereas colistin and mitomycin C retained this activity. Gentamicin exhibited concentration-dependent bactericidal efficacy against resource-depleted cells; specifically, at concentrations >153 the minimum inhibitory concentration (MIC) (Figure S1A), we observed significant bacterial cell killing under nutrient-depleted conditions (Figure 1C).…”

“…We note here that while nutrient availability is an effective means through which bacterial metabolic state can be modulated, previous work has shown that treatment of cells with bacteriostatic antibiotics also suppresses metabolic activity (Lobritz et al, 2015;Lin et al, 2014), which inhibits further killing by bactericidal antibiotics from the b-lactam, quinolone, and aminoglycoside classes (Lobritz et al, 2015;McCall et al, 2019). Indeed, we confirmed that a 30-min pre-treatment with the bacteriostatic agent chloramphenicol protected E. coli from killing by ampicillin, ciprofloxacin, and gentamicin, but not from colistin and mitomycin C (Figures S2G-S2K), consistent with our observations of bacterial cell killing by these antibiotics in media of varying richness.…”

“…Furthermore, it has been demonstrated that the efficacy of such antibiotics against bacterial persisters necessitates co-administration of carbon sources for metabolic activation (Allison et al, 2011;Meylan et al, 2018). Interestingly, however, there are examples of molecules not within these common classes that retain bactericidal efficacy against metabolically repressed cells, suggesting that functionally discrete bactericidal antibiotics have varying metabolic requirements for bacterial cell killing (McCall et al, 2019;Grassi et al, 2017;Kwan et al, 2015;Chowdhury et al, 2016;Eng et al, 1991;Conlon et al, 2013;Defraine et al, 2018). Examples of compounds that retain bactericidal efficacy against persister cells include colistin (McCall et al, 2019;Grassi et al, 2017) and the DNA cross-linking anticancer drug mitomycin C (Kwan et al, 2015).…”

“…Safe and effective methods to eradicate bacteria regardless of metabolic state are critically needed. Currently, however, the direct comparison of metabolic dependencies between discrete antibiotic classes is limited by varying experimental conditions across studies (McCall et al, 2019;Grassi et al, 2017;Kwan et al, 2015;Chowdhury et al, 2016;Eng et al, 1991;Conlon et al, 2013). Here, we investigate the metabolic dependencies of various bactericidal antibiotic classes under standardized conditions.…”

Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant Staphylococcus aureus Under Daptomycin Exposure

“…This investigation aimed to face the analysis of DAP-R mechanisms in MRSA strains on the basis of the acquired knowledge and considering new aspects. First, the "already known" DAP-R mechanisms (dlt over-expression, mprF mutations, and increased net positive cell-surface charge) do not always correlate with changes in DAP minimum inhibitory concentrations (MICs) (Mishra et al, 2014); second, during an infection, the bacteria elapse long periods in limited/arrested growth as in the late growth phases (Kolter et al, 1993); third, DAP is also active on non-dividing and not metabolically active bacteria (Mascio et al, 2007;McCall et al, 2019).…”