Antibiotic tolerance, persistence, and resistance of the evolved minimal cell, Mycoplasma mycoides JCVI-Syn3B

Hossain, Tahmina; Deter, Heather S.; Peters, Eliza J.; Butzin, Nicholas C.

doi:10.1016/j.isci.2021.102391

Cited by 21 publications

(18 citation statements)

References 101 publications

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“…We did a similar FT with clones grown from a single cell and treated them with a lethal Amp concentration (0.1 mg/mL). % Survival was determined for 1 h and 3 h of treatment using persister assays 26,27,34 , and short-term tolerance at 1 h does not indicate the level of persistence at 3 h with lethal Amp; no correlation at 1 h vs 3 h population (r 2 = 0.02). If the primed cells are advantageous for long-term survival, we expect the high persister populations observed at 3 h treatment to stay high with more prolonged antibiotic exposure.…”

Section: Resultsmentioning

confidence: 99%

Escherichia coli cells are primed for survival before lethal antibiotic stress

Hossain

Singh

Butzin

2022

Preprint

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Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population, referred to as persister cells, to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically altered state. One fundamental question is whether this heterogeneous persister population is due to a pre-existing genetic mutation or a result of a transiently-primed reversible cell state. To explore this, we tested clonal populations starting from a single cell using a modified Luria–Delbrück fluctuation test. Through we kept the conditions the same, the diversity in persistence level among clones was relatively consistent: varying from ~ 60–100 and ~ 40–70 fold for ampicillin (Amp) and apramycin (Apr), respectively. Then we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for ~ 5 generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (> 7 generations). Our result demonstrates 1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers, 2) the primed memory state is reproducible and transient, passed down for generations but eventually lost, and 3) a heterogeneous persister population is a result of a transiently-primed reversible cell state and not due to a pre-existing genetic mutation.

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Section: Resultsmentioning

confidence: 99%

Escherichia coli cells are primed for survival before lethal antibiotic stress

Hossain

Singh

Butzin

2022

Preprint

View full text Add to dashboard Cite

show abstract

“…We did a similar FT with clones grown from a single cell and treated them with a lethal Amp concentration (0.1 mg/mL). % Survival was determined for 1 h and 3 h of treatment using persister assays 26, 27, 34 , and short-term tolerance at 1 h does not indicate the level of persistence at 3 h with lethal Amp; no correlation at 1 h vs 3 h population (r 2 = 0.02). If the primed cells are advantageous for long-term survival, we expect the high persister populations observed at 3 h treatment to stay high with more prolonged antibiotic exposure.…”

Section: Resultsmentioning

confidence: 99%

Escherichia colicells are primed for survival before lethal antibiotic stress

Hossain

Singh

Butzin

2022

Preprint

Self Cite

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Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population, referred to as persister cells, to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically altered state. One fundamental question is whether this heterogeneous persister population is due to a pre-existing genetic mutation or a result of a transiently-primed reversible cell state. To explore this, we tested clonal populations starting from a single cell using a modified Luria-Delbruck fluctuation test. Through we kept the conditions the same, the diversity in persistence level among clones was relatively consistent: varying from ~60-100 and ~40-70 fold for ampicillin (Amp) and apramycin (Apr), respectively. Then we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for ~5 generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (>7 generations). Our result demonstrates 1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers, 2) the primed memory state is reproducible and transient, passed down for generations but eventually lost, and 3) a heterogeneous persister population is a result of a transiently-primed reversible cell state and not due to a pre-existing genetic mutation.

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“…neoformans, the results of this study showed that Fluc-O2ME-NP outperformed free Fluc at both tested pH conditions, especially at pH 5. Fungal cells incubated with Fluc alone, even at the highest tested drug concentration of 175 μg/mL, demonstrated a plateau behavior indicating antifungal resistance with a cell density of around 10–25% persisting from 10 to 24 h with no indication of fungal cell populations reducing . On the contrary, C.…”

Section: Discussionmentioning

confidence: 96%

Application of Fluconazole-Loaded pH-Sensitive Lipid Nanoparticles for Enhanced Antifungal Therapy

Rajesh

Gangadoo

Nguyen

et al. 2022

ACS Appl. Mater. Interfaces

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Cryptococcus neoformans is a yeast-like fungus that can cause the life-threatening disease cryptococcal meningitis. Numerous reports have shown increased resistance of this fungus against antifungal treatments, such as fluconazole (Fluc), contributing to an 80% global mortality rate. This work presents a novel approach to improve the delivery of the antifungal agent Fluc and increase the drug's targetability and availability at the infection site. Exploiting the acidic environment surrounding a C. neoformans infected site, we have developed pHsensitive lipid nanoparticles (LNP) encapsulating Fluc to inhibit the growth of resistant C. neoformans. The LNP-Fluc delivery system consists of a neutral lipid monoolein (MO) and a novel synthetic ionizable lipid 2-morpholinoethyl oleate (O2ME). At neutral pH, because of the presence of O2ME, the nanoparticles are neutral and exhibit a liquid crystalline hexagonal nanostructure (hexosomes). At an acidic pH, they are positively charged with a cubic nanostructure (cubosomes), which facilitates the interaction with the negatively charged fungal cell wall. This interaction results in the MIC50 and MIC90 values of the LNP-Fluc being significantly lower than that of the free-Fluc control. Confocal laser scanning microscopy and scanning electron microscopy further support the MIC values, showing fungal cells exposed to LNP-Fluc at acidic pH were heavily distorted, demonstrating efflux of cytoplasmic molecules. In contrast, fungal cells exposed to Fluc alone showed cell walls mostly intact. This current study represents a significant advancement in delivering targeted antifungal therapy to combat fungal antimicrobial resistance.

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Antibiotic tolerance, persistence, and resistance of the evolved minimal cell, Mycoplasma mycoides JCVI-Syn3B

Cited by 21 publications

References 101 publications

Escherichia coli cells are primed for survival before lethal antibiotic stress

Escherichia coli cells are primed for survival before lethal antibiotic stress

Escherichia colicells are primed for survival before lethal antibiotic stress

Application of Fluconazole-Loaded pH-Sensitive Lipid Nanoparticles for Enhanced Antifungal Therapy

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