AIM： To study the effects of disruption of sarA gene on biofilm formation and antibiotic resistance of Staphylococcus epidermidis （ S. epiderrnidis）. METHODS： In order to disrupt sarA gene, the double- crossover h...AIM： To study the effects of disruption of sarA gene on biofilm formation and antibiotic resistance of Staphylococcus epidermidis （ S. epiderrnidis）. METHODS： In order to disrupt sarA gene, the double- crossover homologous recombination was applied in S. epiderrnidis RP62A, and tetracycline resistance gene （tet） was used as the selective marker which was amplified by PCR from the pBR322 and inserted into the locus between sarA upstream and downstream, resulting in pBT2ΔsarA. By electroporation, the plasmid pBT2ΔsarA was transformed into S. epiderrnidis. Gene transcription was detected by real-time reverse transcription-PCR （RT-PCR）. Determination of biofilm was performed in 96-well flat-bottomed culture plates, and antibiotic resistance was analyzed with test tube culture by spectrophotometry at 570 nm respectively. RESULTS： A sarA disrupted strain named S. epiderrnidis RP62AΔsarA was constructed, which was completely defective in biofilm formation, while the sarA complement strain RP62AΔsarA （pHPS9sarA） restored the biofilm formation phenotype. Additionally, the knockout of sarA resulted in decreased erythromycin and kanamycin resistance of S. epiderrnidis RP62A. Compared to the original strain, S. epiderrnidis RP62AΔsarA had an increase of the sensitivity to erythromycin at 200-400 μg/mL and kanamycin at 200-800 μg/mL respectively. CONCLUSION： The knockout of sarA can result in the defect in biofilm formation and the decreased erythromycin and kanamycin resistance in S. epiderrnidis RP62A.展开更多
Biofilm-associated infections are difficult to treat in the clinics because the bacteria embedded in biofilm are ten to thousand times more resistant to traditional antibiotics than planktonic ones.Here,a smart hydrog...Biofilm-associated infections are difficult to treat in the clinics because the bacteria embedded in biofilm are ten to thousand times more resistant to traditional antibiotics than planktonic ones.Here,a smart hydrogel comprised of aminoglycoside antibiotics,pectinase,and oxidized dextran was developed to treat local biofilm-associated infections.The primary amines on aminoglycosides and pectinase were reacted with aldehyde groups on oxidized dextran via a pH-sensitive Schiff base linkage to form the hydrogel.Upon bacterial infection,the increased acidity triggers the release of both pectinase and aminoglycoside antibiotics.The released pectinase efficiently degrades extracellular polysaccharides surrounding the bacteria in biofilm,and thus greatly sensitizes the bacteria to aminoglycosides.The smart hydrogel efficiently eradicated biofilms and killed the embedded bacteria both in vitro and in vivo.This study provides a promising strategy for the treatment of biofilm-associated infections.展开更多
基金Supported by the National Natural Science Foundation of China,No. 30270018
文摘AIM： To study the effects of disruption of sarA gene on biofilm formation and antibiotic resistance of Staphylococcus epidermidis （ S. epiderrnidis）. METHODS： In order to disrupt sarA gene, the double- crossover homologous recombination was applied in S. epiderrnidis RP62A, and tetracycline resistance gene （tet） was used as the selective marker which was amplified by PCR from the pBR322 and inserted into the locus between sarA upstream and downstream, resulting in pBT2ΔsarA. By electroporation, the plasmid pBT2ΔsarA was transformed into S. epiderrnidis. Gene transcription was detected by real-time reverse transcription-PCR （RT-PCR）. Determination of biofilm was performed in 96-well flat-bottomed culture plates, and antibiotic resistance was analyzed with test tube culture by spectrophotometry at 570 nm respectively. RESULTS： A sarA disrupted strain named S. epiderrnidis RP62AΔsarA was constructed, which was completely defective in biofilm formation, while the sarA complement strain RP62AΔsarA （pHPS9sarA） restored the biofilm formation phenotype. Additionally, the knockout of sarA resulted in decreased erythromycin and kanamycin resistance of S. epiderrnidis RP62A. Compared to the original strain, S. epiderrnidis RP62AΔsarA had an increase of the sensitivity to erythromycin at 200-400 μg/mL and kanamycin at 200-800 μg/mL respectively. CONCLUSION： The knockout of sarA can result in the defect in biofilm formation and the decreased erythromycin and kanamycin resistance in S. epiderrnidis RP62A.
基金the National Key R&D Program of ChinaSynthetic Biology Research(2019YFA0904500)+1 种基金the National Natural Science Foundation of China(21725402 and51672191)the Natural Science Foundation of Shanghai(19ZR1415600)。
文摘Biofilm-associated infections are difficult to treat in the clinics because the bacteria embedded in biofilm are ten to thousand times more resistant to traditional antibiotics than planktonic ones.Here,a smart hydrogel comprised of aminoglycoside antibiotics,pectinase,and oxidized dextran was developed to treat local biofilm-associated infections.The primary amines on aminoglycosides and pectinase were reacted with aldehyde groups on oxidized dextran via a pH-sensitive Schiff base linkage to form the hydrogel.Upon bacterial infection,the increased acidity triggers the release of both pectinase and aminoglycoside antibiotics.The released pectinase efficiently degrades extracellular polysaccharides surrounding the bacteria in biofilm,and thus greatly sensitizes the bacteria to aminoglycosides.The smart hydrogel efficiently eradicated biofilms and killed the embedded bacteria both in vitro and in vivo.This study provides a promising strategy for the treatment of biofilm-associated infections.
