Rapid Freezing Enables Aminoglycosides To Eradicate Bacterial Persisters via Enhancing Mechanosensitive Channel MscL-Mediated Antibiotic Uptake

Abstract

Bacterial persisters exhibit noninherited antibiotic tolerance and are linked to the recalcitrance of bacterial infections. It is very urgent but also challenging to develop antipersister strategies. Here, we report that 10-s freezing with liquid nitrogen dramatically enhances the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many Gram-negative pathogens, with weaker potentiation effects on Gram-positive bacteria. In particular, antibiotic-tolerant Escherichia coli and Pseudomonas aeruginosa persisters-which were prepared by treating exponential-phase cells with ampicillin, ofloxacin, the protonophore cyanide m-chlorophenyl hydrazone (CCCP), or bacteriostatic antibiotics-can be effectively killed. We demonstrated, as a proof of concept, that freezing potentiated the aminoglycosides' killing of P. aeruginosa persisters in a mouse acute skin wound model. Mechanistically, freezing dramatically increased the bacterial uptake of aminoglycosides regardless of the presence of CCCP, indicating that the effects are independent of the proton motive force (PMF). In line with these results, we found that the effects were linked to freezing-induced cell membrane damage and were attributable, at least partly, to the mechanosensitive ion channel MscL, which was able to directly mediate such freezing-enhanced aminoglycoside uptake. In view of these results, we propose that the freezing-induced aminoglycoside potentiation is achieved by freezing-induced cell membrane destabilization, which, in turn, activates the MscL channel, which is able to effectively take up aminoglycosides in a PMF-independent manner. Our work may pave the way for the development of antipersister strategies that utilize the same mechanism as freezing but do so without causing any injury to animal cells.

Inactivation of Nitrite-Dependent Nitric Oxide Biosynthesis Is Responsible for Overlapped Antibiotic Resistance between Naturally and Artificially Evolved Pseudomonas aeruginosa

“…The antibiotic bactericidal assay was carried out as previously described ( 32 , 33 ). As mentioned above, after culture in M9 with or without metabolites and/or antibiotics, bacteria were incubated at 37°C with shaking at 200 rpm for 6 h. To determine CFU per ml, 100-μl samples were 10-fold serially diluted and an aliquot of 10 μl of each dilution was spotted onto the LB agar plates and cultured at 37°C for about 11 h. The percent survival was determined by dividing the CFU obtained from the treated sample by the CFU obtained from the control.…”

Synergy of alanine and gentamicin to reduce nitric oxide for elevating killing efficacy to antibiotic-resistant Vibrio alginolyticus

“…Antibiotic bactericidal assay was carried out as described previously [ 17 ]. The well-cultured bacterial cells described above were collected and washed three times and then suspended in M9 with 10 mM acetate, 2 mM MgSO 4 and 100 mM CaCl 2 to 0.6 of OD 600 nm.…”

Escherichia coliCryptic Prophages Sense Nutrients to Control Persister Cell Resuscitation

“…To determine whether cryptic prophages affect persister cell formation, we converted the wild-type, the Δ9 strain with all nine cryptic prophages deleted 2 , and each single cryptic prophage mutant 2 to persister cells using a rifampicin pretreatment 9 and enumerated them. This method of generating persister cells has been validated eight ways 19 and utilized by at least 15 independent labs to date 9,20,[23][24][25][26][27][28][29][30][31][32][33][34][35] ; the approach has the benefit of increasing the number of persister cells by 10,000-fold 9 which enables single-cell studies 3,5,15,19 . We found the presence of the cryptic prophages had no significant impact on the number of persister cells formed with ampicillin (Fig.…”