Freezing Potentiates Aminoglycosides Against Bacterial Persisters

Dormancy is a universal survival strategy for organisms to cope with unfavorable environmental conditions.It is closely related to basic life activities such as development,aging and stress responses.Organisms in dormant state are highly resistant to environmental stresses.The study on dormancy can help us understand how organisms can withstand harsh environments and maintain recovery.Bacteria survive in a complex environment,and in order to cope with the threat of harsh environmental changes,a clever dormancy mechanism has evolved in bacteria.Pathogens in the dormant period can tolerate antibiotic treatment,which largely accounts for the repeated infections and the emergence of broad-spectrum resistant strains.It is an urgent demand in clinic to develop effective strategies for killing the antibiotic-tolerant dormant pathogens.However,it is difficult to kill dormant bacterial cells because antibiotic molecules in a conventional treatment are unable to efficiently enter dormant bacterial cells or the target of antibiotics is inactivated during dormancy.In this study,we found that aminoglycoside antibiotics in combination with rapid freezing could kill the model bacterium Escherichia coli and its persisters that are tolerant to antibiotic treatment.Further studies revealed that this combined treatment could kill some Gram-negative pathogens,such as Pseudomonas aeruginosa,Pseudomonas hydrophila,Shigella,Salmonella,etc.,but it was only moderately effective for a small number of Gram-positive conditional pathogenic bacteria.The effectiveness of the treatment was verified in the mouse model of acute Epidermal wound infection,although mice had freezing injury at certain degree.Further,we found that the combined treatment in a short time(around 10 seconds)could effectively kill the growing and dormant bacteria,and its efficiency was influenced by the freezing rate and the thawing rate after freezing.Importantly,the efficiency of the combined treatment was found to be independent of the proton motive force,which is distinct from the general view that aminoglycoside antibiotics must rely on the proton motive force generated by the membrane potential to enter the bacteria.The bacteria treated with the combination of freezing and aminoglycoside antibiotics increased the uptake of antibiotics.This increase seems to be due to the fact that freezing facilitates aminoglycoside molecules to transport across the bacterial cell membrane in a short time,possibly through some mechano-sensitive ion channels.In addition,we found that upon freezing the integrity of bacterial cell membrane was damaged and bacterial intracellular proteins were leaked out to certain degree.In this study,the utilization of co-treatment of freezing and aminoglycoside antibiotics can effectively kill persisters,which provide a theoretical basis for the development of tolerant bacterial eradication strategy.