Abstract
Bacterial persistence due to antibiotic tolerance is a critical aspect of antibiotic treatment failure, disease latency, and chronic or reemergent infections. The levels of persisters is especially notable for the opportunistic Gram-negative pathogens from the Burkholderia and Pseudomonas genera. We examined the rate of drug tolerant persisters in Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia cepacia complex organisms, and Pseudomonas aeruginosa at mid-log growth in LB broth culture. We found that a fraction of the antibiotic-sensitive cells from every species were tolerant to a 24 h high-dose antibiotic challenge. All tested Burkholderia strains demonstrated a drug tolerant persister population at a rate that was at least 100–500 times higher than P. aeruginosa. When challenged with at least a 10X minimum inhibitory concentration (MIC) 24 h exposure to three different antibiotics with different modes of action we found that in B. pseudomallei Bp82 each of the tree antibiotics revealed different persister fractions at each of two different growth states. This observation suggests that our assay is detecting heterogeneous persister subpopulations. Persistence in B. pseudomallei Bp82 was highly dependent on growth stage, with a surprisingly high persister fraction of >64% of the late stationary phase cells being antibiotic tolerant to 100XMIC cefotaxime. Adaptation of B. pseudomallei to distilled water storage resulted in a population of drug tolerant cells up to 100% of the non-drug-challenged viable cell count in the same cefotaxime assay. Cultivation of B. pseudomallei with a sub-inhibitory concentration of several antibiotics resulted in altered persister fractions within the population relative to cultures lacking the antibiotic. Our study provides insight into the sensitivity of the persister fraction within the population of B. pseudomallei due to environmental variables and suggests diversity within the persister population revealed by different challenge antibiotics.
Highlights
The emergence of resistant organisms was reported soon after the introduction of antibiotics into clinical practice (Abraham and Chain, 1940)
Based on previous studies in Burkholderia spp. and Pseudomonas aeruginosa of disease recurrence (Drevinek and Mahenthiralingam, 2011) and extreme long term viability under nutrient restriction including distilled water for B. pseudomallei (Moore et al, 2008; Hamad et al, 2011), we hypothesized that members of the Burkholderia genus compared to Pseudomonas aeruginosa PA14 were likely to enter a persister state within a population of in vitro growing cells at mid-log phase growth at a high measurable frequency
In the absence of cell wall synthesis in a dormant persister cell a β-lactam antibiotic will not kill the cell since the killing is dependent upon inhibition of the cell wall synthesis during cellular replication
Summary
The emergence of resistant organisms was reported soon after the introduction of antibiotics into clinical practice (Abraham and Chain, 1940). Burkholderia pseudomallei Persisters survives a lethal antibiotic challenge is termed persistence or drug tolerance. This phenomenon is distinct from resistance in that once these drug tolerant cells resume normal growth after the drug is removed; they exhibit the same drug sensitivity as the population originally treated. For reviews of persistence see (Lewis, 2010; Cohen et al, 2013). Increasing evidence suggests that drug tolerance or persistence is an actively maintained state, triggered and enabled by a network of intracellular stress responses that result in drug tolerance in a fraction of the microbial isogenic population (Lewis, 2010)
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