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Freezing enhances the bactericidal action of aminoglycosides against both stationary-phase and exponential-phase E. coli cells. (A) Survival of E. coli stationary-phase cells following a 10-s treatment consisting of freezing in liquid nitrogen plus indicated aminoglycosides, with the treatment being cycled one, two, or three times. (B) Survival of E. coli stationary-phase cells following 10-s, 1-min, or 3-min treatment of freezing plus tobramycin. (C) Survival of E. coli exponential-phase cells following 10-s treatment of freezing plus indicated antibiotics. Tob, tobramycin; Strep, streptomycin; Genta, gentamicin; Kana, kanamycin; Amp, ampicillin; Ofl, ofloxacin. The treatment concentrations of the antibiotics are described in Table S1B.
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Antibiotics have long been used to successfully kill bacterial pathogens, but antibiotic resistance/tolerance usually has led to the failure of antibiotic therapy, and it has become a severe threat to human health. How to improve the efficacy of existing antibiotics is of importance for combating antibiotic-resistant/tolerant pathogens. Here, we re...
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... we noticed that stationary-phase E. coli cells showed significantly reduced viability after the aminoglycoside-containing bacterial cultures were frozen in liquid nitrogen (196°C; Fig. 1A) for 10 s or in ethanol prechilled at 80°C (see Fig. S1A in the supplemental material) for 20 s and subsequently thawed in an ice-water bath. When such combined treatments were repeated two or three times, cell viability was further reduced ( Fig. 1A; see also Fig. S1A). Notably, such potentiation by freezing appears to be specific for ...
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... we noticed that stationary-phase E. coli cells showed significantly reduced viability after the aminoglycoside-containing bacterial cultures were frozen in liquid nitrogen (196°C; Fig. 1A) for 10 s or in ethanol prechilled at 80°C (see Fig. S1A in the supplemental material) for 20 s and subsequently thawed in an ice-water bath. When such combined treatments were repeated two or three times, cell viability was further reduced ( Fig. 1A; see also Fig. S1A). Notably, such potentiation by freezing appears to be specific for aminoglycosides, without influencing two other classes ...
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... after the aminoglycoside-containing bacterial cultures were frozen in liquid nitrogen (196°C; Fig. 1A) for 10 s or in ethanol prechilled at 80°C (see Fig. S1A in the supplemental material) for 20 s and subsequently thawed in an ice-water bath. When such combined treatments were repeated two or three times, cell viability was further reduced ( Fig. 1A; see also Fig. S1A). Notably, such potentiation by freezing appears to be specific for aminoglycosides, without influencing two other classes of bactericidal antibiotics, i.e., -lactams (ampicillin and carbenicillin) and fluoroquinolones (ofloxacin Survival of E. coli stationary-phase cells following a 10-s treatment consisting of ...
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... of freezing plus tobramycin. (C) Survival of E. coli exponential-phase cells following 10-s treatment of freezing plus indicated antibiotics. Tob, tobramycin; Strep, streptomycin; Genta, gentamicin; Kana, kanamycin; Amp, ampicillin; Ofl, ofloxacin. The treatment concentrations of the antibiotics are described in Table S1B. and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also ...
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... antibiotics. Tob, tobramycin; Strep, streptomycin; Genta, gentamicin; Kana, kanamycin; Amp, ampicillin; Ofl, ofloxacin. The treatment concentrations of the antibiotics are described in Table S1B. and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or ...
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... Strep, streptomycin; Genta, gentamicin; Kana, kanamycin; Amp, ampicillin; Ofl, ofloxacin. The treatment concentrations of the antibiotics are described in Table S1B. and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or warming rather than during the ...
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... and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or warming rather than during the frozen ...
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... (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or warming rather than during the frozen ...
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... further support of the aminoglycoside potentiation by freezing, stationary-phase E. coli cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of ...
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... further support of the aminoglycoside potentiation by freezing, stationary-phase E. coli cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate ...
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... E. coli cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate antibiotic-tolerant E. coli persisters or persister-like cells in a PMF-independent manner. Bacterial ...
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... cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate antibiotic-tolerant E. coli persisters or persister-like cells in a PMF-independent manner. Bacterial persisters are widely ...
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... cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate antibiotic-tolerant E. coli persisters or persister-like cells in a PMF-independent manner. Bacterial persisters are widely believed to account for recurrent infections and antibiotic resistance development (5, 7-9, 31), and their eradication is of particular clinical interest. We ...
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... major findings in our study include (i) the potentiation effect of rapid freezing on aminoglycoside action against both normal bacterial cells and antibiotic-tolerant persisters ( Fig. 1, 2, and 4; see also Fig. S6 in the supplemental material) (Table 1) and (ii) a direct role of the mechanosensitive ion channel MscL in mediating such potentiation ( Fig. 8 and 9; see also Fig. S9). Not surprisingly, the potentiation effect is achieved by enhancing the bacterial uptake of aminoglycosides. Remarkably, this freezingenhanced ...
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... we propose that the freezing-enhanced aminoglycoside uptake is most likely accomplished by cytoplasmic membrane-localized ion channels such as MscL (as diagrammed in the left part of Fig. 10), based on several independent observations. First, E. coli mscL cells exhibited a marginal increase in their resistance to the combined treatment (Fig. S8E). Second, only the complementary expression of the MscL channel, but not that of MscS, MscK, and YbdG channels, dramatically The key to the potentiation of aminoglycosides by ...
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... MscL is able to nonspecifically transport ions to a certain degree (77), it is conceivable that Ca 2 and Mg 2 can also be transported into cells by freezing-activated MscL. As such, high (up to 10 mM) concentrations of Ca 2 and Mg 2 in cell suspensions would be able to competitively inhibit MscL-mediated aminoglycoside uptake (as diagrammed in Fig. 10) at a concentration of aminoglycoside of around 100 ...
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... possible explanation for the freezing-induced aminoglycoside potentiation is that crevices or pores are formed in the cytoplasmic membrane of bacterial cells during the freezing/thawing treatment, such that aminoglycosides are able to freely diffuse through these crevices (as illustrated in the bottom right part of Fig. 10). This hypothesis is supported by the observations that freezing leads to (i) leakage of a large amount of cellular proteins ( Fig. 7B) and other intracellular content (56-59) from the cells, (ii) increased permeability of the cell membrane ( Fig. 7C; see also Fig. S8A), and (iii) cell membrane bending and wrinkles (Fig. 7D). ...
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... freezing has been utilized to treat skin, prostate, lung, and other types of cancers (43-46) (termed cryotherapy or cryosurgery). To the best of our knowledge, our study is the first report of its application in treating bacterial infections, possibly because the bactericidal effects of freezing alone on bacterial pathogens are weak ( Fig. 1 and 4; see also Fig. S5) (71). A direct application of our combined treatment protocol to the eradication of persister bacterial cells would obviously be limited due to the inevitable freezing injury to animal cells and tissues. ...
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... been utilized to treat skin, prostate, lung, and other types of cancers (43-46) (termed cryotherapy or cryosurgery). To the best of our knowledge, our study is the first report of its application in treating bacterial infections, possibly because the bactericidal effects of freezing alone on bacterial pathogens are weak ( Fig. 1 and 4; see also Fig. S5) (71). A direct application of our combined treatment protocol to the eradication of persister bacterial cells would obviously be limited due to the inevitable freezing injury to animal cells and tissues. Nonetheless, our work is still of interest for developing promising antipersister strategies, based on the following ...
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... we noticed that stationary-phase E. coli cells showed significantly reduced viability after the aminoglycoside-containing bacterial cultures were frozen in liquid nitrogen (196°C; Fig. 1A) for 10 s or in ethanol prechilled at 80°C (see Fig. S1A in the supplemental material) for 20 s and subsequently thawed in an ice-water bath. When such combined treatments were repeated two or three times, cell viability was further reduced ( Fig. 1A; see also Fig. S1A). Notably, such potentiation by freezing appears to be specific for ...
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... we noticed that stationary-phase E. coli cells showed significantly reduced viability after the aminoglycoside-containing bacterial cultures were frozen in liquid nitrogen (196°C; Fig. 1A) for 10 s or in ethanol prechilled at 80°C (see Fig. S1A in the supplemental material) for 20 s and subsequently thawed in an ice-water bath. When such combined treatments were repeated two or three times, cell viability was further reduced ( Fig. 1A; see also Fig. S1A). Notably, such potentiation by freezing appears to be specific for aminoglycosides, without influencing two other classes ...
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... after the aminoglycoside-containing bacterial cultures were frozen in liquid nitrogen (196°C; Fig. 1A) for 10 s or in ethanol prechilled at 80°C (see Fig. S1A in the supplemental material) for 20 s and subsequently thawed in an ice-water bath. When such combined treatments were repeated two or three times, cell viability was further reduced ( Fig. 1A; see also Fig. S1A). Notably, such potentiation by freezing appears to be specific for aminoglycosides, without influencing two other classes of bactericidal antibiotics, i.e., -lactams (ampicillin and carbenicillin) and fluoroquinolones (ofloxacin Survival of E. coli stationary-phase cells following a 10-s treatment consisting of ...
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... January/February 2020 Volume 11 Issue 1 e03239-19 mbio.asm.org 3 and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also ...
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... January/February 2020 Volume 11 Issue 1 e03239-19 mbio.asm.org 3 and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or ...
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... January/February 2020 Volume 11 Issue 1 e03239-19 mbio.asm.org 3 and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or warming rather than during the ...
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... 3 and ciprofloxacin) (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or warming rather than during the frozen state. In further support of the aminoglycoside potentiation by freezing, stationary-phase E. coli cells largely survived after a 3-h treatment with ...
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... (Fig. S1B). Neither repeated freezing nor antibiotic treatment alone for 30 min at room temperature significantly killed the cells (Fig. 1A, top panel; see also Fig. S1A). In addition, extension of the duration of the combined treatment of tobramycin and freezing from 10 s to 3 min did not further reduce bacterial viability ( Fig. 1B; see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or warming rather than during the frozen state. In further support of the aminoglycoside potentiation by freezing, stationary-phase E. coli cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. ...
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... see also Fig. S1C), implying that the molecular events accounting for such aminoglycoside potentiation take place during cooling and/or warming rather than during the frozen state. In further support of the aminoglycoside potentiation by freezing, stationary-phase E. coli cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of ...
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... rather than during the frozen state. In further support of the aminoglycoside potentiation by freezing, stationary-phase E. coli cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate ...
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... E. coli cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate antibiotic-tolerant E. coli persisters or persister-like cells in a PMF-independent manner. Bacterial ...
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... cells largely survived after a 3-h treatment with aminoglycoside at 37°C (Fig. S1D, line 1 versus line 2 [except for streptomycin]); however, these surviving cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate antibiotic-tolerant E. coli persisters or persister-like cells in a PMF-independent manner. Bacterial persisters are widely ...
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... cells were effectively killed upon subsequent cycled freezing (Fig. S1D, line 2 versus lines 3 to 5). We also found that freezing performed only once, either at 196°C (Fig. 1C) or at 80°C (Fig. S1E), was able to dramatically enhance the bactericidal action of aminoglycosides against exponential-phase E. coli cells (lines 4, 6, 8, and 10 in Fig. 1C; see also Freezing enables aminoglycosides to eradicate antibiotic-tolerant E. coli persisters or persister-like cells in a PMF-independent manner. Bacterial persisters are widely believed to account for recurrent infections and antibiotic resistance development (5, 7-9, 31), and their eradication is of particular clinical interest. We ...
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... major findings in our study include (i) the potentiation effect of rapid freezing on aminoglycoside action against both normal bacterial cells and antibiotic-tolerant persisters ( Fig. 1, 2, and 4; see also Fig. S6 in the supplemental material) (Table 1) and (ii) a direct role of the mechanosensitive ion channel MscL in mediating such potentiation ( Fig. 8 and 9; see also Fig. S9). Not surprisingly, the potentiation effect is achieved by enhancing the bacterial uptake of aminoglycosides. Remarkably, this freezingenhanced ...
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... we propose that the freezing-enhanced aminoglycoside uptake is most likely accomplished by cytoplasmic membrane-localized ion channels such as MscL (as diagrammed in the left part of Fig. 10), based on several independent observations. First, E. coli mscL cells exhibited a marginal increase in their resistance to the combined treatment (Fig. S8E). Second, only the complementary expression of the MscL channel, but not that of MscS, MscK, and YbdG channels, dramatically The key to the potentiation of aminoglycosides by ...
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... MscL is able to nonspecifically transport ions to a certain degree (77), it is conceivable that Ca 2 and Mg 2 can also be transported into cells by freezing-activated MscL. As such, high (up to 10 mM) concentrations of Ca 2 and Mg 2 in cell suspensions would be able to competitively inhibit MscL-mediated aminoglycoside uptake (as diagrammed in Fig. 10) at a concentration of aminoglycoside of around 100 ...
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... possible explanation for the freezing-induced aminoglycoside potentiation is that crevices or pores are formed in the cytoplasmic membrane of bacterial cells during the freezing/thawing treatment, such that aminoglycosides are able to freely diffuse through these crevices (as illustrated in the bottom right part of Fig. 10). This hypothesis is supported by the observations that freezing leads to (i) leakage of a large amount of cellular proteins ( Fig. 7B) and other intracellular content (56-59) from the cells, (ii) increased permeability of the cell membrane ( Fig. 7C; see also Fig. S8A), and (iii) cell membrane bending and wrinkles (Fig. 7D). ...
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... freezing has been utilized to treat skin, prostate, lung, and other types of cancers (43-46) (termed cryotherapy or cryosurgery). To the best of our knowledge, our study is the first report of its application in treating bacterial infections, possibly because the bactericidal effects of freezing alone on bacterial pathogens are weak ( Fig. 1 and 4; see also Fig. S5) (71). A direct application of our combined treatment protocol to the eradication of persister bacterial cells would obviously be limited due to the inevitable freezing injury to animal cells and tissues. ...
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... been utilized to treat skin, prostate, lung, and other types of cancers (43-46) (termed cryotherapy or cryosurgery). To the best of our knowledge, our study is the first report of its application in treating bacterial infections, possibly because the bactericidal effects of freezing alone on bacterial pathogens are weak ( Fig. 1 and 4; see also Fig. S5) (71). A direct application of our combined treatment protocol to the eradication of persister bacterial cells would obviously be limited due to the inevitable freezing injury to animal cells and tissues. Nonetheless, our work is still of interest for developing promising antipersister strategies, based on the following ...
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Citations
... Studies have shown that mechanosensitive channels play a decisive role in antibiotic sensitivity (12,15). In order to investigate the role of MscS and MscL in antibiotic resistance of A. pleuropneumoniae, we performed antibiotic susceptibility tests on six strains. ...
Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae . In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo . In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.
IMPORTANCE
Bacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae , while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae . MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae .
... The detection of cell membrane permeability with propidium iodide (PI) staining was performed as described before with slight modifications. 40 CRPA-3 at logarithmic phase was incubated with AS101 or/and mefloquine (AS101 32 μg/mL; mefloquine 64 μg/mL) at 37 °C for 2 h, and the bacteria in the non-drug group served as the control group. ...
Background
In recent years, carbapenem-resistant Pseudomonas aeruginosa (CRPA) has spread around the world, leading to a high mortality and close attention of medical community. In this study, we aim to find a new strategy of treatment for CRPA infections.
Methods
Eight strains of CRPA were collected, and PCR detected the multi-locus sequence typing (MLST). The antimicrobial susceptibility test was conducted using the VITEK@2 compact system. The minimum inhibitory concentration (MIC) for AS101 and mefloquine was determined using the broth dilution method. Antibacterial activity was tested in vitro and in vivo through the chessboard assay, time killing assay, and a mouse model. The mechanism of AS101 combined with mefloquine against CRPA was assessed through the biofilm formation inhibition assay, electron microscopy, and detection of reactive oxygen species (ROS).
Results
The results demonstrated that all tested CRPA strains exhibited multidrug resistance. Moreover, our investigation revealed a substantial synergistic antibacterial effect of AS101-mefloquine in vitro. The assay for inhibiting biofilm formation indicated that AS101-mefloquine effectively suppressed the biofilm formation of CRPA-5 and CRPA-6. Furthermore, AS101-mefloquine were observed to disrupt the bacterial cell wall and enhance the permeability of the cell membrane. This effect was achieved by stimulating the production of ROS, which in turn hindered the growth of CRPA-3. To evaluate the therapeutic potential, a murine model of CRPA-3 peritoneal infection was established. Notably, AS101-mefloquine administration resulted in a significant reduction in bacterial load within the liver, kidney, and spleen of mice after 72 hours of treatment.
Conclusion
The present study showed that the combination of AS101 and mefloquine yielded a notable synergistic bacteriostatic effect both in vitro and in vivo, suggesting a potential clinical application of this combination in the treatment of CRPA.
... Some of the mechanisms by which MscL influences antibiotic sensitivity have been studied only recently [25]. Changes in MS channel expression, when combined with changes in osmolarity [38], cell wall integrity via ampicillin treatment [39,40], or even freezing [41], have been shown to influence antimicrobial susceptibility. Thus, it may not be too surprising that changes in specific MS channel expression, when combined with aerosolization, also influence antibiotic resistance. ...
Highlights
What are the main findings? Aerosolization and ventilation air velocity affect antibiotic resistance of E. coli strains;
Bacteria respond to ventilated environments through mechanosensitive ion channels triggered by aerosolization;
What is the implication of the main finding? Critical infrastructures require real-time knowledge about their environment for microbiome source tracking;
Clinical indoor spaces where bacterial infections are treated can result in potential exposure to bioaerosols.
Abstract
Understanding how bacteria respond to ventilated environments is a crucial concept, especially when considering accurate airflow modeling and detection limits. To properly design facilities for aseptic conditions, we must minimize the parameters for pathogenic bacteria to thrive. Identifying how pathogenic bacteria continue to survive, particularly due to their multi-drug resistance characteristics, is necessary for designing sterile environments and minimizing pathogen exposure. A conserved characteristic among bacterial organisms is their ability to maintain intracellular homeostasis for survival and growth in hostile environments. Mechanosensitive (MS) channels are one of the characteristics that guide this phenomenon. Interestingly, during extreme stress, bacteria will forgo favorable homeostasis to execute fast-acting survival strategies. Physiological sensors, such as MS channels, that trigger this survival mechanism are not clearly understood, leaving a gap in how bacteria translate physical stress to an intracellular response. In this paper, we study the role of mechanosensitive ion channels that are potentially triggered by aerosolization. We hypothesize that change in antimicrobial uptake is affected by aerosolization stress. Bacteria regulate their defense mechanisms against antimicrobials, which leads to varying susceptibility. Based on this information we hypothesize that aerosolization stress affects the antimicrobial resistance defense mechanisms of Escherichia coli (E. coli). We analyzed the culturability of knockout E. coli strains with different numbers of mechanosensitive channels and compared antibiotic susceptibility under stressed and unstressed airflow conditions. As a result of this study, we can identify how the defensive mechanisms of resistant bacteria are triggered for their survival in built environments. By changing ventilation airflow velocity and observing the change in antibiotic responses, we show how pathogenic bacteria respond to ventilated environments via mechanosensitive ion channels.
... Arguably, the overall microbial population remaining in the apical part of the root canal (i.e., that from the pulverised specimens in this study) would be more important and relevant where post-treatment disease is concerned, as the irrigant solution would have been removed and the canal dried before progressing to the next step in a clinical situation. On the other hand, the liquid nitrogen used in the pulverisation procedure may have a bacteriocidal enhancing effect that confounded the result obtained [44], casting doubt on the accuracy of this sampling method in bacteriological evaluation. Furthermore, it is unknown if the pulverisation process may cause any disruption to the integrity and viability of bacterial cells, casting doubt on the validity of pulverisation as a microbial sampling method in endodontics. ...
Purpose:
This study aimed to compare the antibacterial effectiveness of passive ultrasonic irrigation (PUI), Er,Cr:YSGG laser (WTL), and photon-induced photoacoustic streaming (PIPS) using an Er:YAG laser against Enterococcus faecalis biofilms in the apical third of root canals.
Methods:
Root canals of 70 single-rooted human teeth were instrumented and infected with E. faecalis for 3 weeks to form biofilms. The samples were randomly divided into five groups as follows: (i) PUI + 3% NaOCl (n = 16); (ii) Er,Cr:YSGG laser (n = 16); (iii) PIPS + 3% NaOCl (n = 16); (iv) positive control group (n = 10); and (v) negative control group (n = 10). The bacterial content in the root canal was sampled using (a) the paper-point sampling method before (S1) and after (S2) treatment and (b) pulverising the apical 5 mm of the root. The number of bacteria recovered from each group was counted as colony-forming units (CFUs). The amount of reduction between the groups was compared with the Kruskal-Wallis test and post-test Dunn's multiple comparisons tests. The significance level was set at 5% (p < 0.05).
Results:
The samples from the paper-point sampling method showed that the amount of bacteria before (S1) and after treatment (S2) was significantly different between PIPS and WTL, as well as between the PUI and WTL groups. In contrast, no significant difference was found between the PIPS and PUI groups. From the pulverised samples, the results indicated no significant difference among all experimental groups in the amount of bacterial reduction in the apical 5 mm of the root.
Conclusions:
PUI and PIPS showed a significantly greater reduction in bacterial content within the main root canal compared with the WTL. There was no difference among all experimental groups in the apical third of the root.
... Falghoush et al. found that osmotic compounds could potentiate typical antibiotics against Acinetobacter baumannii biofilms (21). We previously observed that hypoionic shock (i.e., treatment with ion-free solutions) and rapid freezing dramatically potentiate aminoglycosides against bacterial persisters by activating mechanosensitive channels and enhancing antibiotic uptake (22)(23)(24)(25)(26). In addition, photothermal materials have been developed to potentiate various antibiotics by imposing chemical and physical effects (27). ...
... Sublethal Heat Shock Enhances the Bactericidal Action of Aminoglycosides against Both Stationary-and Exponential-Phase E. coli Cells. We previously reported that rapid freezing (e.g., 10-s freezing with liquid nitrogen) and 1-min hypoionic shock remarkably potentiate aminoglycoside in killing E. coli cells (22,23,26). We thus sought to examine whether other physical stress conditions could do the same. ...
... the combined treatment using tobramycin of different concentrations plus heat shock at 50 °C (SI Appendix, Fig. S4B). By contrast, our recently developed aminoglycoside potentiation approaches using rapid freezing or hypoionic shock failed to facilitate tobramycin to kill these multidrug-resistant pathogens (23,26). Given these, we further determined minimal inhibitory concentrations (MICs) of these pathogens to tobramycin, which were up to around 16,000 μg/mL (Fig. 3B). ...
The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli. This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae. Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.
... After DHS causes effluxes of potassium and glutamate via the open MscL, the drug then uses the open pore to pass into the cytoplasm [54]. Consistent with these findings, there is also recent evidence that MscL is gated upon cell freezing, which can induce aminoglycoside uptake and potentiation [56]. MscL is likely a pathway to the cytoplasm for other antibiotics [53]. ...
The drug-resistance crisis has become dire and new antibiotic targets and strategies are required. MscL is a conserved bacterial mechanosensitive channel that plays the role of “osmotic-emergency-release-valve”. It has the largest gated pore known allowing osmoprotectants out, and other compounds into the cell. Inappropriate gating of the channel can lead to slowed growth, decreased viability, and an increased in potency for many antibiotics. The “membrane permeability” observed for some antibiotics, including streptomycin, is mediated by directly binding to and activating MscL. Novel compounds that are MscL agonists have also recently been isolated. Although the compounds are diverse, the binding sites of all characterized MscL-specific agonists are within the same general region of the MscL complex, leading to an in silico screening for compounds that bind this region. In sum, these studies demonstrate that MscL is a viable drug target that may lead to a new generation of antibiotics and adjuvants.
... Some physical treatments have also been shown to increase aminoglycoside uptake and susceptibility. Physical treatments such as rapid freezing in liquid nitrogen have been shown to increase aminoglycoside uptake by a PMF independent route through membrane disruption in E. coli and P. aeruginosa antibiotic tolerant persister cells (Zhao et al. 2020). Although successful in a mouse skin wound model (Zhao et al. 2020) there is the obvious application problems of damage to host cells caused by the freezing process, combined with the inability to treat systemic infections. ...
... Physical treatments such as rapid freezing in liquid nitrogen have been shown to increase aminoglycoside uptake by a PMF independent route through membrane disruption in E. coli and P. aeruginosa antibiotic tolerant persister cells (Zhao et al. 2020). Although successful in a mouse skin wound model (Zhao et al. 2020) there is the obvious application problems of damage to host cells caused by the freezing process, combined with the inability to treat systemic infections. ...
... In vivo efficacy, dosage, off target side effects, route of administration and toxicity all need to be considered. Some potential adjuvants such as rhamnolipids (Radlinski et al. 2019) or physical treatments such as rapid freezing (Zhao et al. 2020) can cause direct damage to human cells. Aminoglycosides can also induce side effects in host cells such as ototoxicity (Fu et al. 2021) which need to be considered when discussing potential adjuvants. ...
Following the discovery of streptomycin from Streptomyces griseus in the 1940s by Selman Waksman and colleagues, aminoglycosides were first used to treat tuberculosis and then numerous derivatives have since been used to combat a wide variety of bacterial infections. These bactericidal antibiotics were used as first-line treatments for several decades but were largely replaced by ß-lactams and fluoroquinolones in the 1980s, although widespread emergence of antibiotic-resistance has led to renewed interest in aminoglycosides. The primary site of action for aminoglycosides is the 30 S ribosomal subunit where they disrupt protein translation, which contributes to widespread cellular damage through a number of secondary effects including rapid uptake of aminoglycosides via elevated proton-motive force (PMF), membrane damage and breakdown, oxidative stress, and hyperpolarisation of the membrane. Several factors associated with aminoglycoside entry have been shown to impact upon bacterial killing, and more recent work has revealed a complex relationship between metabolic states and the efficacy of different aminoglycosides. Hence, it is imperative to consider the environmental conditions and bacterial physiology and how this can impact upon aminoglycoside entry and potency. This mini-review seeks to discuss recent advances in this area and how this might affect the future use of aminoglycosides.
... The discovery and development of new antibiotics, including structurally modified existing antibiotics, has played a dominant role in this war (Brown & Wright, 2016;Lewis, 2020). In addition, improving the efficacy of existing antibiotics has also attracted attentions in the recent decade (Allison, Brynildsen & Collins, 2011;Peng et al., 2015;Liu et al., 2019;Wright, 2016;Zhao et al., 2020;Lv et al., 2022a;Lv et al., 2022b), given that these existing antibiotics have been well documented in their toxicity, pharmacokinetics, administration and mechanism of actions. ...
... Previously, we found that indole and 5-methylindole could markedly potentiate aminoglycoside antibiotics against bacterial persister cells under hypoionic conditions (i.e., in ion-free solutions) (Sun et al., 2020) after short-term combined treatment. Although such potentiation could be fulfilled within 5-min incubation, the requirement of hypoionic conditions for treatment may limit the potential of its application, as salts and electrolytes are ubiquitously present throughout human and animal bodies. ...
... To this end, nutrient shift-induced S. aureus persisters were prepared by switching the carbon source of S. aureus cells in exponential-phase to fumarate (Radzikowski et al., 2016), and starvation-induced S. aureus persisters were made by transferring S. aureus cells in stationary-phase to medium without any nutrients (Chen et al., 2019). These nutrient shift-induced and starvation-induced S. aureus persister cells were kept in non-replicating status and highly tolerant to the attack by tobramycin under conventional treatment condition ( Fig. S1; or refer to our earlier reports (Sun et al., 2020;Chen et al., 2019)). However, both of them were killed by 5-methylindole and 2methylindole in a concentration-dependent manner, with indole being much less effective (Figs. ...
Antibiotic resistance of bacterial pathogens has become a severe threat to human health. To counteract antibiotic resistance, it is of significance to discover new antibiotics and also improve the efficacy of existing antibiotics. Here we show that 5-methylindole, a derivative of the interspecies signaling molecule indole, is able to directly kill various Gram-positive pathogens ( e.g ., Staphylococcus aureus and Enterococcus faecalis ) and also Gram-negative ones ( e.g ., Escherichia coli and Pseudomonas aeruginosa ), with 2-methylindole being less potent. Particularly, 5-methylindole can kill methicillin-resistant S. aureus , multidrug-resistant Klebsiella pneumoniae , Mycobacterium tuberculosis , and antibiotic-tolerant S. aureus persisters. Furthermore, 5-methylindole significantly potentiates aminoglycoside antibiotics, but not fluoroquinolones, killing of S. aureus . In addition, 5-iodoindole also potentiates aminoglycosides. Our findings open a new avenue to develop indole derivatives like 5-methylindole as antibacterial agents or adjuvants of aminoglycoside.
... Next, 100-mL samples were serially diluted 10-fold and spread onto LB agar to determine bacterial survival. For liquid nitrogen stimulation (38), 1 mL of logarithmic-phase bacteria was collected, frozen in liquid nitrogen for 30 s, and then thawed at 37°C in a 5% CO 2 incubator. The survival of bacteria was also determined by the plate count method. ...
A. baumannii was identified as a top-priority pathogen by the WHO due to its antibiotic resistance. Meanwhile, the pathogenicity of A. baumannii mediated by several vital virulence factors also cannot be ignored.
... Antibiotic bactericidal assay. As previously described, antibiotic bactericidal assay was performed in M9 with or without metabolites or/and antibiotics and incubated at 37°C with 200 rpm (40). Six hours later, 100 mL of samples were 10-fold continuously diluted and 10 mL of each dilution was aliquoted on the 2% LB agar plates and cultured at 37°C for about 11 h, and CFU per mL was determined. ...
Antibiotic-resistant Pseudomonas aeruginosa has become a real concern in hospital-acquired infections, especially in critically ill and immunocompromised patients. Understanding antibiotic resistance mechanisms and developing novel control measures are highly appreciated.
